Human-derived bacteria that induce proliferation or accumulation of regulatory T cells

ABSTRACT

Species of human-derived bacteria belonging to the Clostridia class have been shown to induce accumulation of regulatory T cells (Treg cells) in the colon and suppress immune functions. Pharmaceutical compositions containing these bacteria can be used to prevent and treat immune-mediated diseases such as autoimmune diseases.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/362,097, filed May 30, 2014, which is a national stage filing under35 U.S.C. § 371 of International Application PCT/JP2012/007687, filedNov. 29, 2012, which claims the benefit under 35 U.S.C. 119(e) of U.S.Provisional Application No. 61/565,976, filed Dec. 1, 2011 and U.S.Provisional Application No. 61/607,360, filed Mar. 6, 2012. The entireteachings of the referenced applications are incorporated herein byreference.

TECHNICAL FIELD

The subject matter described herein relates to a composition ofhuman-derived bacteria that induces proliferation, accumulation, orproliferation and accumulation of regulatory T cells and whichcomprises, as an active component, (a) one or more (a, at least one)human-derived bacteria that belongs to the Clostridia class, (b) culturesupernatant of one or more (a, at least one) of the bacteria; (c) aphysiologically active substance derived from one or more of thebacteria or (d) a combination of any two or more of the foregoing. Italso relates to a method for inducing proliferation, accumulation orproliferation and accumulation of regulatory T cells. The composition,which comprises any of (a)-(d) above, is referred to as a bacterialcomposition. Moreover, the subject matter relates to a method fortreating or preventing at least one disease or condition that isresponsive to induction of regulatory T cells, such as autoimmunediseases, inflammatory diseases, and infectious diseases, byadministering the bacterial composition to an individual in needthereof.

BACKGROUND

Hundreds of species of commensal microorganisms are harbored in thegastrointestinal tracts of mammals, where they interact with the hostimmune system. Research using germ-free (GF) animals has shown that thecommensal microorganisms influence the development of the mucosal immunesystem, such as histogenesis of Peyer's patches (PPs) and isolatedlymphoid follicles (ILFs), secretion of antimicrobial peptides from theepithelium, and accumulation of unique lymphocytes in mucosal tissues,including immunoglobulin A-producing plasma cells, intraepitheliallymphocytes, IL-17-producing CD4-positive T cells (Th 17), andIL-22-producing NK-like cells (Non-Patent Literature (NPL) 1 to 7).Consequently, the presence of intestinal bacteria enhances protectivefunctions of the mucous membranes, enabling the host to mount robustimmune responses against pathogenic microbes invading the body. On theother hand, the mucosal immune system maintains unresponsiveness todietary antigens and harmless microbes (NPL Document 3). Abnormality inthe regulation of cross-talk between commensal bacteria and the immunesystem (intestinal dysbiosis) may lead to overly robust immune responseto environmental antigens and inflammatory bowel disease (IBD) mayresult (NPL 8 to 10).

Recent studies have shown that individual commensal bacteria controldifferentiation of their specific immune cells in the mucosal immunesystem. For example, Bacteroides fragilis, which is a commensalbacterium in humans, specifically induces a systemic Th1 cell responseand a mucosal IL-10-producing T cell response in mice, and plays a rolein protecting the host from colitis, which is caused by a pathogen (NPL3). Segmented filamentous bacteria, which are intestinal commensalbacteria in mice, induce mucosal Th17 cell response and enhanceresistance against infection of gastrointestinal tracts of the host witha pathogen (NPL 11 to 13). In addition, short-chain fatty acids derivedfrom several commensal bacteria are known to suppress intestinalinflammation (NPL 14). Moreover, it has been observed that the presenceof some species of intestinal microbiota greatly influences thedifferentiation of regulatory T cells (hereafter referred to as “Tregcells”) which help maintain homeostasis of the immune system. Althoughspecific species of murine bacterial commensals that can stronglystimulate Tregs have been identified (NPL 15), it is still unknownwhether species of human commensal bacteria exert an equivalentinfluence on the human immune system. Furthermore, the human intestinaltract harbors more than a thousand bacterial species, many of which havenot yet been cultured (NPL 16). It is not feasible to guess a prioriwhich ones, if any, might have an effect on Tregs.

In order to develop drugs, dietary supplements, or foods with beneficialimmune functions for human use, it is desirable to identify commensalmicroorganisms that naturally colonize humans and have immune-modulatingproperties. Furthermore, since many of the commensals in the humanmicrobiome have yet to be cultured, it is necessary to develop methodsto cultivate them so that they can be produced by traditional industrialfermentation processes and subsequently incorporated in pharmaceuticalor food formulations.

CD4⁺ T cells are regulatory T cells that have been identified as a cellsubset that suppresses immunity. A transcription factor, Foxp3, isexpressed in CD4⁺ T cells, which are known to play an important role inmaintaining immunological homeostasis (NPL 8, 9, 17, and 18).Foxp3-expressing cells are present in large numbers in the colon andonly Treg cells present locally in the colon constantly express IL-10,an immunosuppressive cytokine, at a high level (NPL 19). Animals havingCD4⁺ Foxp3⁺ cells from which IL-10 is specifically removed developinflammatory bowel disease (NPL 20).

Accordingly, there is a need to identify human-derived commensalbacterial species with the ability to strongly induce Treg cells toproduce IL-10 in the colon at a high level and to develop methods toculture such species. Such species could be used to enhanceimmunosuppression, which, in turn, can be applied to treatment ofautoimmune diseases, such as inflammatory bowel disease, inflammatorydiseases, allergies, or organ transplantation, among other diseases andconditions.

NON PATENT LITERATURE

-   [NPL 1] J. J. Cebra, “Am J Clin Nutr”, May, 1999, 69, 1046S-   [NPL 2] A. J. Macpherson, N. L. Harris, “Nat Rev Immunol”, June    2004, 4, 478-   [NPL 3] J. L. Round, S. K. Mazmanian, “Nat Rev Immunol”, May 2009,    9, 313-   [NPL 4] D. Bouskra et al., “Nature”, Nov. 27, 2008, 456, 507-   [NPL 5] K. Atarashi et al., “Nature”, Oct. 9, 2008, 455, 808-   [NPL 6] Ivanov, II et al., “Cell Host Microbe”, Oct. 16, 2008, 4,    337-   [NPL 7] S. L. Sanos et al., “Nat Immunol”, January 2009, 10, 83-   [NPL 8] M. A. Curotto de Lafaille, J. J. Lafaille, “Immunity”, May    2009, 30, 626-   [NPL 9] M. J. Barnes, F. Powrie, “Immunity”, Sep. 18, 2009, 31, 401-   [NPL 10] W. S. Garrett et al., “Cell”, Oct. 5, 2007, 131, 33-   [NPL 11] Ivanov, II et al., “Cell”, Oct. 30, 2009, 139, 485.-   [NPL 12] V. Gaboriau-Routhiau et al., “Immunity”, Oct. 16, 2009, 31,    677-   [NPL 13] N. H. Salzman et al., “Nat Immunol”, 11, 76.-   [NPL 14] K. M. Maslowski et al., “Nature”, Oct. 29, 2009, 461, 1282-   [NPL 15] K. Atarashi et al., “Science”, Jan. 21, 2011, 331, 337-   [NPL 16] J. Quin et al., “Nature”, Mar. 4, 2010, 464, 59-   [NPL 17] L. F. Lu, A. Rudensky, “Genes Dev”, Jun. 1, 2009, 23, 1270-   [NPL 18] S. Sakaguchi, T. Yamaguchi, T. Nomura, M. Ono, “Cell”, May    30, 2008, 133, 775-   [NPL 19] C. L. Maynard et al., “Nat Immunol”, September 2007, 8, 931-   [NPL 20] Y. P. Rubtsov et al., “Immunity”, April 2008, 28, 546

SUMMARY

The present compositions and methods have been made in view of theabove-described problems in the art. Described herein are methods ofidentifying and culturing intestinal commensal bacteria, isolated fromhumans, which induce, preferably strongly induce, the proliferation,accumulation, or proliferation and accumulation of regulatory T cells.Described are compositions, also referred to as bacterial compositions,that (1) comprise (a) one or more of the identified intestinal commensal(human-derived) bacteria; (b) a culture supernatant of one or more ofthe bacteria; (c) one or more physiologically active substance derivedfrom one or more of the bacteria or from one or more of the culturesupernatant; (d) or a combination of any two or three of (a)-(c) and (2)induce the proliferation and/or accumulation of regulatory T cells (Tregcells). Alternatively, a composition comprises (a) one or more of theidentified intestinal commensal (human-derived) bacteria; (b) a culturesupernatant of one or more of the bacteria; or (c) one or morephysiologically active substance derived from the bacteria or from theculture supernatant, wherein the composition induces proliferationand/or accumulation of regulatory T cells. In some embodiments, thecomposition comprises one or more of the identified intestinal commensal(human-derived) bacteria. In some embodiments, the composition comprisesa culture supernatant of one or more of the bacteria. In someembodiments, the composition comprises one or more physiologicallyactive substance derived from the bacteria or from the culturesupernatant. In some embodiments, the one or more bacteria or one ormore physiologically active substance derived from the bacteria is threeor more. In some embodiments, the one or more bacteria or one or morephysiologically active substance derived from the bacteria is five ormore. In some embodiments, the one or more bacteria or one or morephysiologically active substance derived from the bacteria is seventeenor more. In some embodiments, the one or more bacteria or one or morephysiologically active substance derived from the bacteria istwenty-three or more. In some embodiments, the one or more bacteria orone or more physiologically active substance derived from the bacteriais 23. In specific embodiments, the bacterial compositions induce, andpreferably strongly induce, proliferation, accumulation, orproliferation and accumulation of regulatory T cells that produce animmunosuppressive cytokine, such as IL-10, in the colon (e.g., the humancolon) at high levels. Such bacterial compositions are useful, forexample, to enhance immunosuppression and, as a result, to treatautoimmune diseases. Bacterial compositions comprise, as an activecomponent, at least one organism and/or at least one substance selectedfrom the group consisting of: Clostridium saccharogumia, Clostridiumramosum JCM1298, Clostridium ramosum, Flavonifractor plautii,Pseudoflavonifractor capillosus ATCC 29799, Clostridium hathewayi,Clostridium saccharolyticum WM1, Bacteroides sp. MANG, Clostridiumsaccharolyticum, Clostridium scindens, Lachnospiraceae bacterium5_1_57FAA, Lachnospiraceae bacterium 6_1_63FAA, Clostridium sp. 14616,Clostridium bolteae ATCC BAA-613, cf. Clostridium sp. MLG055,Erysipelotrichaceae bacterium 2_2_44A, Clostridium indolis, Anaerostipescaccae, Clostridium bolteae, Lachnospiraceae bacterium DJF_VP30,Lachnospiraceae bacterium 3_1_57FAA_CT1, Anaerotruncus colihominis,Anaerotruncus colihominis DSM 17241, Ruminococcus sp. ID8,Lachnospiraceae bacterium 2_1_46FAA, Clostridium lavalense, Clostridiumasparagiforme DSM 15981, Clostridium symbiosum, Clostridium symbiosumWAL-14163, Eubacterium contortum, Clostridium sp. D5, Oscillospiraceaebacterium NML 061048, Oscillibacter valericigenes, Lachnospiraceaebacterium A4, Clostridium sp. 316002/08, and Clostridiales bacterium1_7_47FAA, Blautia cocoides, Anaerostipes caccae DSM 14662; a culturesupernatant of at least one (a, one or more) of the bacteriadescribed/listed herein; a physiologically active substance derived from(a, one or more) bacteria described/listed herein or any combination oftwo or three of the foregoing. Alternatively, bacterial compositionscomprise, as an active component, at least one organism or at least onesubstance selected from the group consisting of: Clostridiumsaccharogumia, Clostridium ramosum JCM1298, Clostridium ramosum,Flavonifractor plautii, Pseudoflavonifractor capillosus ATCC 29799,Clostridium hathewayi, Clostridium saccharolyticum WM1, Bacteroides sp.MANG, Clostridium saccharolyticum, Clostridium scindens, Lachnospiraceaebacterium 5_1_57FAA, Lachnospiraceae bacterium 6_1_63FAA, Clostridiumsp. 14616, Clostridium bolteae ATCC BAA-613, cf. Clostridium sp. MLG055,Erysipelotrichaceae bacterium 2_2_44A, Clostridium indolis, Anaerostipescaccae, Clostridium bolteae, Lachnospiraceae bacterium DJF_VP30,Lachnospiraceae bacterium 3_1_57FAA_CT1, Anaerotruncus colihominis,Anaerotruncus colihominis DSM 17241, Ruminococcus sp. ID8,Lachnospiraceae bacterium 2_1_46FAA, Clostridium lavalense, Clostridiumasparagiforme DSM 15981, Clostridium symbiosum, Clostridium symbiosumWAL-14163, Eubacterium contortum, Clostridium sp. D5, Oscillospiraceaebacterium NML 061048, Oscillibacter valericigenes, Lachnospiraceaebacterium A4, Clostridium sp. 316002/08, and Clostridiales bacterium1_7_47FAA, Blautia cocoides, Anaerostipes caccae DSM 14662; a culturesupernatant of at least one (a, one or more) of the bacteriadescribed/listed herein; a physiologically active substance derived from(a, one or more) bacteria described/listed herein. In some embodiments,a bacterial composition comprises at least one organism selected fromthe group consisting of: Clostridium saccharogumia, Clostridium ramosumJCM1298, Clostridium ramosum, Flavonifractor plautii,Pseudoflavonifractor capillosus ATCC 29799, Clostridium hathewayi,Clostridium saccharolyticum WM1, Bacteroides sp. MANG, Clostridiumsaccharolyticum, Clostridium scindens, Lachnospiraceae bacterium5_1_57FAA, Lachnospiraceae bacterium 6_1_63FAA, Clostridium sp. 14616,Clostridium bolteae ATCC BAA-613, cf. Clostridium sp. MLG055,Erysipelotrichaceae bacterium 2_2_44A, Clostridium indolis, Anaerostipescaccae, Clostridium bolteae, Lachnospiraceae bacterium DJF_VP30,Lachnospiraceae bacterium 3_1_57FAA_CT1, Anaerotruncus colihominis,Anaerotruncus colihominis DSM 17241, Ruminococcus sp. ID8,Lachnospiraceae bacterium 2_1_46FAA, Clostridium lavalense, Clostridiumasparagiforme DSM 15981, Clostridium symbiosum, Clostridium symbiosumWAL-14163, Eubacterium contortum, Clostridium sp. D5, Oscillospiraceaebacterium NML 061048, Oscillibacter valericigenes, Lachnospiraceaebacterium A4, Clostridium sp. 316002/08, and Clostridiales bacterium1_7_47FAA, Blautia cocoides, Anaerostipes caccae DSM 14662. In someembodiments, a bacterial composition comprises a culture supernatant ofat least one (a, one or more) of the bacteria described/listed herein.In some embodiments, a bacterial composition comprises a physiologicallyactive substance derived from (a, one or more) bacteria described/listedherein. In some embodiments, the one or more bacteria or one or morephysiologically active substance derived from the bacteria is three ormore. In some embodiments, the one or more bacteria or one or morephysiologically active substance derived from the bacteria is five ormore. In some embodiments, the one or more bacteria or one or morephysiologically active substance derived from the bacteria is 17 ormore. In some embodiments, the one or more bacteria or one or morephysiologically active substance derived from the bacteria is 23 ormore. In some embodiments, the one or more bacteria or one or morephysiologically active substance derived from the bacteria is 23.Bacterial compositions can comprise any bacteria (Clostridia or otherbacteria) that contain DNA comprising a nucleotide sequence havingsufficient homology with sequences provided herein and that exhibitsubstantially the same effect on regulatory T cells as that exerted byany one of the following: Clostridium saccharogumia, Clostridium ramosumJCM1298, Clostridium ramosum, Flavonifractor plautii,Pseudoflavonifractor capillosus ATCC 29799, Clostridium hathewayi,Clostridium saccharolyticum WM1, Bacteroides sp. MANG, Clostridiumsaccharolyticum, Clostridium scindens, Lachnospiraceae bacterium5_1_57FAA, Lachnospiraceae bacterium 6_1_63FAA, Clostridium sp. 14616,Clostridium bolteae ATCC BAA-613, cf. Clostridium sp. MLG055,Erysipelotrichaceae bacterium 2_2_44A, Clostridium indolis, Anaerostipescaccae, Clostridium bolteae, Lachnospiraceae bacterium DJF_VP30,Lachnospiraceae bacterium 3_1_57FAA_CT1, Anaerotruncus colihominis,Anaerotruncus colihominis DSM 17241, Ruminococcus sp. ID8,Lachnospiraceae bacterium 2_1_46FAA, Clostridium lavalense, Clostridiumasparagiforme DSM 15981, Clostridium symbiosum, Clostridium symbiosumWAL-14163, Eubacterium contortum, Clostridium sp. D5, Oscillospiraceaebacterium NML 061048, Oscillibacter valericigenes, Lachnospiraceaebacterium A4, Clostridium sp. 316002/08, and Clostridiales bacterium1_7_47FAA, Blautia cocoides, and Anaerostipes caccae DSM 14662. In someembodiments, bacteria present in bacterial compositions have at least90% (90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) homologywith sequences provided herein, such as, but not limited to, thenucleotide sequences designated OTU herein and listed, for example, atthe pages following the last Example. In specific embodiments, suchbacteria contain DNA comprising a nucleotide sequence that has at least90% (90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) homologywith one or more DNA sequence designated herein as follows: OTU136;OTU46; OTU221; OTU9; OTU296; OTU21; OTU166; OTU73; OTU174; OTU14; OTU55;OTU337; OTU314; OTU195; OTU306; OTU87; OTU86; OTU152; OTU253; OTU259;OTU281; OTU288; OTU334; OTU359; OTU362; or OTU367. Alternatively,bacteria contain DNA comprising a nucleotide sequence that has at least90% (90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) homologywith DNA of one or more of the following: Clostridium saccharogumia,Clostridium ramosum JCM1298, Clostridium ramosum, Flavonifractorplautii, Pseudoflavonifractor capillosus ATCC 29799, Clostridiumhathewayi, Clostridium saccharolyticum WM1, Bacteroides sp. MANG,Clostridium saccharolyticum, Clostridium scindens, Lachnospiraceaebacterium 5_1_57FAA, Lachnospiraceae bacterium 6_1_63FAA, Clostridiumsp. 14616, Clostridium bolteae ATCC BAA-613, cf. Clostridium sp. MLG055,Erysipelotrichaceae bacterium 2_2_44A, Clostridium indolis, Anaerostipescaccae, Clostridium bolteae, Lachnospiraceae bacterium DJF_VP30,Lachnospiraceae bacterium 3_1_57FAA_CT1, Anaerotruncus colihominis,Anaerotruncus colihominis DSM 17241, Ruminococcus sp. ID8,Lachnospiraceae bacterium 2_1_46FAA, Clostridium lavalense, Clostridiumasparagiforme DSM 15981, Clostridium symbiosum, Clostridium symbiosumWAL-14163, Eubacterium contortum, Clostridium sp. D5, Oscillospiraceaebacterium NML 061048, Oscillibacter valericigenes, Lachnospiraceaebacterium A4, Clostridium sp. 316002/08, and Clostridiales bacterium1_7_47FAA, Blautia cocoides, and Anaerostipes caccae DSM 14662. Inspecific embodiments, bacterial compositions comprise bacteria (such ashuman-derived bacteria) that contain DNA comprising a nucleotidesequence having at least 97%, 98% or 99% homology with sequencesprovided herein, such as, but not limited to, the nucleotide sequencesdesignated OTU herein and listed, for example, at the pages followingthe last Example. In specific embodiments, the bacteria in bacterialcompositions contain DNA comprising a nucleotide sequence that has atleast 97%, 98% or 99% homology with one or more DNA sequence designatedherein as follows: OTU136; OTU46; OTU221; OTU9; OTU296; OTU21; OTU166;OTU73; OTU174; OTU14; OTU55; OTU337; OTU314; OTU195; OTU306; OTU87;OTU86; OTU152; OTU253; OTU259; OTU281; OTU288; OTU334; OTU359; OTU362;or OTU367. Alternatively, the bacteria contain DNA comprising anucleotide sequence that has at least 97%, 98% or 99% homology with DNAof one or more of the following: Clostridium saccharogumia, Clostridiumramosum JCM1298, Clostridium ramosum, Flavonifractor plautii,Pseudoflavonifractor capillosus ATCC 29799, Clostridium hathewayi,Clostridium saccharolyticum WM1, Bacteroides sp. MANG, Clostridiumsaccharolyticum, Clostridium scindens, Lachnospiraceae bacterium5_1_57FAA, Lachnospiraceae bacterium 6_1_63FAA, Clostridium sp. 14616,Clostridium bolteae ATCC BAA-613, cf. Clostridium sp. MLG055,Erysipelotrichaceae bacterium 2_2_44A, Clostridium indolis, Anaerostipescaccae, Clostridium bolteae, Lachnospiraceae bacterium DJF_VP30,Lachnospiraceae bacterium 3_1_57FAA_CT1, Anaerotruncus colihominis,Anaerotruncus colihominis DSM 17241, Ruminococcus sp. ID8,Lachnospiraceae bacterium 2_1_46FAA, Clostridium lavalense, Clostridiumasparagiforme DSM 15981, Clostridium symbiosum, Clostridium symbiosumWAL-14163, Eubacterium contortum, Clostridium sp. D5, Oscillospiraceaebacterium NML 061048, Oscillibacter valericigenes, Lachnospiraceaebacterium A4, Clostridium sp. 316002/08, and Clostridiales bacterium1_7_47FAA, Blautia cocoides, Anaerostipes caccae DSM 14662. Any of thebacteria of the Clostridia class can be present in spore form orvegetative form.

Solution of Problem

As described herein, among the more than a thousand species of bacteriain the human microbiome, there are several species that strongly inducethe accumulation of Tregs in the colon. As also described, although mostbacterial species present in fecal samples from healthy individuals donot have the ability to stimulate Tregs, species that belong to theClostridia class have the ability to cause a robust induction of Tregsin the colon. Moreover, the inventors have obtained in vitro cultures ofeach of the bacterial species identified and shown that inoculating micewith the in vitro cultured species also leads to a robust accumulationof Tregs in the colon.

As described herein, compositions that comprise, as an active component,(a) one or more of certain species of bacteria that belong to theClostridia class or bacteria that contain DNA comprising a nucleotidesequence having at least 90% homology with sequences provided herein, inspore form or in vegetative form; (b) a culture supernatant of one ormore such bacteria; (c) one or more physiologically active substancederived from (a) or (b); or (d) a combination of any two or three of(a), (b) and (c) and induce the proliferation and/or accumulation ofregulatory T cells (Treg cells) suppress immune functions.

More specifically:

One embodiment is a composition that induces proliferation, accumulationor both proliferation and accumulation of regulatory T cells, thecomposition comprising, as an active component, at least one organismand/or at least one substance selected from the group consisting of:Clostridium saccharogumia, Clostridium ramosum JCM1298, Clostridiumramosum, Flavonifractor plautii, Pseudoflavonifractor capillosus ATCC29799, Clostridium hathewayi, Clostridium saccharolyticum WM1,Bacteroides sp. MANG, Clostridium saccharolyticum, Clostridium scindens,Lachnospiraceae bacterium 5_1_57FAA, Lachnospiraceae bacterium6_1_63FAA, Clostridium sp. 14616, Clostridium bolteae ATCC BAA-613, cf.Clostridium sp. MLG055, Erysipelotrichaceae bacterium 2_2_44A,Clostridium indolis, Anaerostipes caccae, Clostridium bolteae,Lachnospiraceae bacterium DJF_VP30, Lachnospiraceae bacterium3_1_57FAA_CT1, Anaerotruncus colihominis, Anaerotruncus colihominis DSM17241, Ruminococcus sp. ID8, Lachnospiraceae bacterium 2_1_46FAA,Clostridium lavalense, Clostridium asparagiforme DSM 15981, Clostridiumsymbiosum, Clostridium symbiosum WAL-14163, Eubacterium contortum,Clostridium sp. D5, Oscillospiraceae bacterium NML 061048, Oscillibactervalericigenes, Lachnospiraceae bacterium A4, Clostridium sp. 316002/08,and Clostridiales bacterium 1_7_47FAA, Blautia cocoides, Anaerostipescaccae DSM 14662; a culture supernatant of at least one of the bacteriadescribed/listed herein, and a physiologically active substance derivedfrom a bacterium described/listed herein.

In some embodiments, the active component is one or more of Clostridiumsaccharogumia, Clostridium ramosum JCM1298, Clostridium ramosum,Flavonifractor plautii, Pseudoflavonifractor capillosus ATCC 29799,Clostridium hathewayi, Clostridium saccharolyticum WM1, Bacteroides sp.MANG, Clostridium saccharolyticum, Clostridium scindens, Lachnospiraceaebacterium 5_1_57FAA, Lachnospiraceae bacterium 6_1_63FAA, Clostridiumsp. 14616, Clostridium bolteae ATCC BAA-613, cf. Clostridium sp. MLG055,Erysipelotrichaceae bacterium 2_2_44A, Clostridium indolis, Anaerostipescaccae, Clostridium bolteae, Lachnospiraceae bacterium DJF_VP30,Lachnospiraceae bacterium 3_1_57FAA_CT1, Anaerotruncus colihominis,Anaerotruncus colihominis DSM 17241, Ruminococcus sp. ID8,Lachnospiraceae bacterium 2_1_46FAA, Clostridium lavalense, Clostridiumasparagiforme DSM 15981, Clostridium symbiosum, Clostridium symbiosumWAL-14163, Eubacterium contortum, Clostridium sp. D5, Oscillospiraceaebacterium NML 061048, Oscillibacter valericigenes, Lachnospiraceaebacterium A4, Clostridium sp. 316002/08, and Clostridiales bacterium1_7_47FAA, Blautia cocoides, Anaerostipes caccae DSM 14662. In someembodiments, the active component is a culture supernatant of one ormore of the bacteria described/listed herein. In some embodiments, theactive component is one or more physiologically active substancesderived from a bacterium described/listed herein. In some embodiments,the one or more bacteria or one or more physiologically active substancederived from the bacteria is three or more. In some embodiments, the oneor more bacteria or one or more physiologically active substance derivedfrom the bacteria is five or more. In some embodiments, the one or morebacteria or one or more physiologically active substance derived fromthe bacteria is 17 or more. In some embodiments, the one or morebacteria or one or more physiologically active substance derived fromthe bacteria is 23 or more. In some embodiments, the one or morebacteria or one or more physiologically active substance derived fromthe bacteria is 23.

A bacterial composition as described herein comprises at least one ofthe following: one bacteria as described herein; at least one culturesupernatant obtained from culture in which one (or more) of the bacteriawas present (grown or maintained) or a fraction of such a supernatant;one or more physiologically active substance derived from one or morebacteria (such as from the bacteria named herein) or a combination ofany two or three of the foregoing. The term composition/bacterialcomposition refers to all such combinations.

The bacteria in the composition can be, for example, Clostridiumsaccharogumia, Clostridium ramosum JCM1298, Clostridium ramosum,Flavonifractor plautii, Pseudoflavonifractor capillosus ATCC 29799,Clostridium hathewayi, Clostridium saccharolyticum WM1, Bacteroides sp.MANG, Clostridium saccharolyticum, Clostridium scindens, Lachnospiraceaebacterium 5_1_57FAA, Lachnospiraceae bacterium 6_1_63FAA, Clostridiumsp. 14616, Clostridium bolteae ATCC BAA-613, cf. Clostridium sp. MLG055,Erysipelotrichaceae bacterium 2_2_44A, Clostridium indolis, Anaerostipescaccae, Clostridium bolteae, Lachnospiraceae bacterium DJF_VP30,Lachnospiraceae bacterium 3_1_57FAA_CT1, Anaerotruncus colihominis,Anaerotruncus colihominis DSM 17241, Ruminococcus sp. ID8,Lachnospiraceae bacterium 2_1_46FAA, Clostridium lavalense, Clostridiumasparagiforme DSM 15981, Clostridium symbiosum, Clostridium symbiosumWAL-14163, Eubacterium contortum, Clostridium sp. D5, Oscillospiraceaebacterium NML 061048, Oscillibacter valericigenes, Lachnospiraceaebacterium A4, Clostridium sp. 316002/08, and Clostridiales bacterium1_7_47FAA, Blautia cocoides, Anaerostipes caccae DSM 14662 or anybacteria (such as human-derived bacteria) that contain DNA comprising atleast 90% homology (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or 100% homology) with sequences provided herein, such as, but notlimited to, the nucleotide sequences designated OTU herein and listed,for example, at the pages following the last Example. In specificembodiments, the bacteria contain DNA comprising a nucleotide sequencethat has at least 97%, at least 98% or at least 99% homology with one ormore DNA sequence designated herein as follows: OTU136; OTU46; OTU221;OTU9; OTU296; OTU21; OTU166; OTU73; OTU174; OTU14; OTU55; OTU337;OTU314; OTU195; OTU306; OTU87; OTU86; OTU152; OTU253; OTU259; OTU281;OTU288; OTU334; OTU359; OTU362; or OTU367. Alternatively, the bacteriacontain DNA comprising a nucleotide sequence that has at least 97% (97%,98%, 99%, 100%) homology with DNA of one or more of the following:Clostridium saccharogumia, Clostridium ramosum JCM1298, Clostridiumramosum, Flavornfractor plautii, Pseudoflavonifractor capillosus ATCC29799, Clostridium hathewayi, Clostridium saccharolyticum WM1,Bacteroides sp. MANG, Clostridium saccharolyticum, Clostridium scindens,Lachnospiraceae bacterium 5_1_57FAA, Lachnospiraceae bacterium6_1_63FAA, Clostridium sp. 14616, Clostridium bolteae ATCC BAA-613, cf.Clostridium sp. MLG055, Erysipelotrichaceae bacterium 2_2_44A,Clostridium indolis, Anaerostipes caccae, Clostridium bolteae,Lachnospiraceae bacterium DJF_VP30, Lachnospiraceae bacterium3_1_57FAA_CT1, Anaerotruncus colihominis, Anaerotruncus colihominis DSM17241, Ruminococcus sp. ID8, Lachnospiraceae bacterium 2_1_46FAA,Clostridium lavalense, Clostridium asparagiforme DSM 15981, Clostridiumsymbiosum, Clostridium symbiosum WAL-14163, Eubacterium contortum,Clostridium sp. D5, Oscillospiraceae bacterium NML 061048, Oscillibactervalericigenes, Lachnospiraceae bacterium A4, Clostridium sp. 316002/08,and Clostridiales bacterium 1_7_47FAA, Blautia cocoides, andAnaerostipes caccae DSM 14662.

In one embodiment, the composition induces regulatory T cells that aretranscription factor Foxp3-positive regulatory T cells orIL-10-producing regulatory T cells. In another embodiment, thecomposition has an immunosuppressive effect.

One embodiment is a pharmaceutical composition that inducesproliferation, accumulation or both proliferation and/or accumulation ofregulatory T cells and suppresses immune function. The pharmaceuticalcomposition comprises a bacterial composition described herein and apharmaceutically acceptable component, such as a carrier, a solvent or adiluent. In specific embodiments, such a pharmaceutical compositioncomprises (a) (1) one or more species of bacteria belonging to theClostridia class, as described herein, in spore form or in vegetativeform, (2) a culture supernatant of such bacteria, (3) a physiologicallyactive substance derived therefrom or (4) a combination of any two orthree of (1), (2) and (3) and (b) a pharmaceutically acceptablecomponent, such as carrier, a solvent or a diluent. In specificembodiments, (a) above is at least one organism or substance selectedfrom the group consisting of: Clostridium saccharogumia, Clostridiumramosum JCM1298, Clostridium ramosum, Flavonifractor plautii,Pseudoflavonifractor capillosus ATCC 29799, Clostridium hathewayi,Clostridium saccharolyticum WM1, Bacteroides sp. MANG, Clostridiumsaccharolyticum, Clostridium scindens, Lachnospiraceae bacterium5_1_57FAA, Lachnospiraceae bacterium 6_1_63FAA, Clostridium sp. 14616,Clostridium bolteae ATCC BAA-613, cf. Clostridium sp. MLG055,Erysipelotrichaceae bacterium 2_2_44A, Clostridium indolis, Anaerostipescaccae, Clostridium bolteae, Lachnospiraceae bacterium DJF_VP30,Lachnospiraceae bacterium 3_1_57FAA_CT1, Anaerotruncus colihominis,Anaerotruncus colihominis DSM 17241, Ruminococcus sp. ID8,Lachnospiraceae bacterium 2_1_46FAA, Clostridium lavalense, Clostridiumasparagiforme DSM 15981, Clostridium symbiosum, Clostridium symbiosumWAL-14163, Eubacterium contortum, Clostridium sp. D5, Oscillospiraceaebacterium NML 061048, Oscillibacter valericigenes, Lachnospiraceaebacterium A4, Clostridium sp. 316002/08, and Clostridiales bacterium1_7_47FAA, Blautia cocoides, Anaerostipes caccae DSM 14662, a culturesupernatant of one or more of the bacteria, and a physiologically activesubstance derived from one or more of the bacteria. In some embodiments,(a) above is at least one organism selected from the group consistingof: Clostridium saccharogumia, Clostridium ramosum JCM1298, Clostridiumramosum, Flavonifractor plautii, Pseudoflavonifractor capillosus ATCC29799, Clostridium hathewayi, Clostridium saccharolyticum WM1,Bacteroides sp. MANG, Clostridium saccharolyticum, Clostridium scindens,Lachnospiraceae bacterium 5_1_57FAA, Lachnospiraceae bacterium6_1_63FAA, Clostridium sp. 14616, Clostridium bolteae ATCC BAA-613, cf.Clostridium sp. MLG055, Erysipelotrichaceae bacterium 2_2_44A,Clostridium indolis, Anaerostipes caccae, Clostridium bolteae,Lachnospiraceae bacterium DJF_VP30, Lachnospiraceae bacterium3_1_57FAA_CT1, Anaerotruncus colihominis, Anaerotruncus colihominis DSM17241, Ruminococcus sp. ID8, Lachnospiraceae bacterium 2_1_46FAA,Clostridium lavalense, Clostridium asparagiforme DSM 15981, Clostridiumsymbiosum, Clostridium symbiosum WAL-14163, Eubacterium contortum,Clostridium sp. D5, Oscillospiraceae bacterium NML 061048, Oscillibactervalericigenes, Lachnospiraceae bacterium A4, Clostridium sp. 316002/08,and Clostridiales bacterium 1_7_47FAA, Blautia cocoides, Anaerostipescaccae DSM 14662. In some embodiments, (1) above is a culturesupernatant of one or more of the bacteria. In some embodiments, (1)above is a physiologically active substance derived from one or more ofthe bacteria. In some embodiments, the at least one organism orsubstances is three or more. In some embodiments, the at least oneorganism or substances is five or more. In some embodiments, the atleast one organism or substances is 17 or more. In some embodiments, theat least one organism or substances is 23 or more. In some embodiments,the at least one organism or substances is 23. In further embodiments,(a)(1) above is bacteria (such as human-derived bacteria) that containDNA comprising at least 90% homology (e.g., 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or 100% homology) with sequences providedherein, such as, but not limited to, the nucleotide sequences designatedOTU herein and listed, for example, at the pages following the lastExample. In specific embodiments of the pharmaceutical composition, thebacteria contain DNA comprising a nucleotide sequence that has at least97%, at least 98% or at least 99% homology with one or more DNA sequencedesignated herein as follows: OTU136; OTU46; OTU221; OTU9; OTU296;OTU21; OTU166; OTU73; OTU174; OTU14; OTU55; OTU337; OTU314; OTU195;OTU306; OTU87; OTU86; OTU152; OTU253; OTU259; OTU281; OTU288; OTU334;OTU359; OTU362; or OTU367. Alternatively, the bacteria in thepharmaceutical composition contain DNA comprising a nucleotide sequencethat has at least 97% (97%, 98%, 99%, 100%) homology with DNA of one ormore of the following: Clostridium saccharogumia, Clostridium ramosumJCM1298, Clostridium ramosum, Flavonifractor plautii,Pseudoflavonifractor capillosus ATCC 29799, Clostridium hathewayi,Clostridium saccharolyticum WM1, Bacteroides sp. MANG, Clostridiumsaccharolyticum, Clostridium scindens, Lachnospiraceae bacterium5_1_57FAA, Lachnospiraceae bacterium 6_1_63FAA, Clostridium sp. 14616,Clostridium bolteae ATCC BAA-613, cf. Clostridium sp. MLG055,Erysipelotrichaceae bacterium 2_2_44A, Clostridium indolis, Anaerostipescaccae, Clostridium bolteae, Lachnospiraceae bacterium DJF_VP30,Lachnospiraceae bacterium 3_1_57FAA_CT1, Anaerotruncus colihominis,Anaerotruncus colihominis DSM 17241, Ruminococcus sp. ID8,Lachnospiraceae bacterium 2_1_46FAA, Clostridium lavalense, Clostridiumasparagiforme DSM 15981, Clostridium symbiosum, Clostridium symbiosumWAL-14163, Eubacterium contortum, Clostridium sp. D5, Oscillospiraceaebacterium NML 061048, Oscillibacter valericigenes, Lachnospiraceaebacterium A4, Clostridium sp. 316002/08, and Clostridiales bacterium1_7_47FAA, Blautia cocoides, Anaerostipes caccae DSM 14662.

The pharmaceutical composition induces the proliferation and/oraccumulation of regulatory T cells (Treg cells) and suppresses immunefunction.

Also provided is a method of inducing proliferation, accumulation orboth proliferation and accumulation of regulatory T cells in anindividual (e.g., an individual in need thereof, such as an individualin need of induction of proliferation and/or accumulation of regulatoryT cells). The method comprises administering to the individual abacterial composition described herein or a pharmaceutical compositioncomprising a bacterial composition described herein. In the method atleast one organism or substance selected from the group consisting of:Clostridium saccharogumia, Clostridium ramosum JCM1298, Clostridiumramosum, Flavonifractor plautii, Pseudoflavonifractor capillosus ATCC29799, Clostridium hathewayi, Clostridium saccharolyticum WM1,Bacteroides sp. MANG, Clostridium saccharolyticum, Clostridium scindens,Lachnospiraceae bacterium 5_1_57FAA, Lachnospiraceae bacterium6_1_63FAA, Clostridium sp. 14616, Clostridium bolteae ATCC BAA-613, cf.Clostridium sp. MLG055, Erysipelotrichaceae bacterium 2_2_44A,Clostridium indolis, Anaerostipes caccae, Clostridium bolteae,Lachnospiraceae bacterium DJF_VP30, Lachnospiraceae bacterium3_1_57FAA_CT1, Anaerotruncus colihominis, Anaerotruncus colihominis DSM17241, Ruminococcus sp. ID8, Lachnospiraceae bacterium 2_1_46FAA,Clostridium lavalense, Clostridium asparagiforme DSM 15981, Clostridiumsymbiosum, Clostridium symbiosum WAL-14163, Eubacterium contortum,Clostridium sp. D5, Oscillospiraceae bacterium NML 061048, Oscillibactervalericigenes, Lachnospiraceae bacterium A4, Clostridium sp. 316002/08,and Clostridiales bacterium 1_7_47FAA, Blautia cocoides, Anaerostipescaccae DSM 14662; a culture supernatant of one or more of the bacteriaor one or more component of the culture supernatant; a physiologicallyactive substance derived from one or more of the bacteria or acombination of two or three of the foregoing is administered to anindividual (also referred to as an individual in need thereof) who canbe a healthy individual or an individual in need of prevention,reduction or treatment of a condition or disease. For example, thecompositions described may be administered to an individual in need oftreatment, reduction in the severity of or prevention of a disease orcondition such as an autoimmune disease, an inflammatory disease, anallergic disease, and an infectious disease

Optionally, administration of the bacterial composition may be incombination with, or preceded by, a course of one or more antibiotics.

Optionally, administration of the bacterial composition may be incombination with administration of at least one prebiotic substance thatpreferentially favors the growth of the species in the bacterialcomposition over the growth of other human commensal bacterial species.In one embodiment, the prebiotic substance(s) is, for example, anondigestible oligosaccharide. In specific embodiments, the one or moreprebiotic substance(s) is selected from the group consisting of almondskin, inulin, oligofructose, raffinose, lactulose, pectin,hemicellulose, amylopectin, acetyl-Co A, biotin, beet molasses, yeastextracts, and resistant starch. Also contemplated herein is acomposition that comprises the bacterial composition and at least oneprebiotic substance.

The bacterial composition may be administered in combination with asubstance selected from the group consisting of corticosteroids,mesalazine, mesalamine, sulfasalazine, sulfasalazine derivatives,immunosuppressive drugs, cyclosporin A, mercaptopurine, azathiopurine,prednisone, methotrexate, antihistamines, glucocorticoids, epinephrine,theophylline, cromolyn sodium, anti-leukotrienes, anti-cholinergic drugsfor rhinitis, anti-cholinergic decongestants, mast-cell stabilizers,monoclonal anti-IgE antibodies, vaccines, anti-TNF inhibitors such asinfliximab, adalimumab, certolizumab pegol, golimumab, or etanercept,and combinations thereof. Also described herein is a composition thatcomprises the bacterial composition and at least one substance selectedfrom the group consisting of corticosteroids, mesalazine, mesalamine,sulfasalazine, sulfasalazine derivatives, immunosuppressive drugs,cyclosporin A, mercaptopurine, azathiopurine, prednisone, methotrexate,antihistamines, glucocorticoids, epinephrine, theophylline, cromolynsodium, anti-leukotrienes, anti-cholinergic drugs for rhinitis,anti-cholinergic decongestants, mast-cell stabilizers, monoclonalanti-IgE antibodies, vaccines, anti-TNF inhibitors such as infliximab,adalimumab, certolizumab pegol, golimumab, or etanercept, andcombinations thereof.

In a further embodiment, the bacterial composition can be used as anadjuvant to improve the efficacy of a vaccine formulation. For example,the bacterial composition can be used as an adjuvant to a vaccine forthe prophylaxis or treatment of an autoimmune disease or an allergicdisease. In some embodiments, a method for prophylaxis or treatment isprovided, the method comprising administering the bacterial compositionand administering a vaccine.

Assessment of the extent of induction of proliferation or accumulationof regulatory T cells that results from administration of a compositiondescribed herein can be carried out by a variety of approaches, such asby measurement of the number of Foxp3-expressing Tregs in a patientsample (such as a biopsy or a blood sample), promotion of IL-10expression, promotion of CTLA4 expression, promotion of IDO expression,suppression of IL-4 expression, or colonization of an individual withthe bacterial composition. The results of such assessments are used asan index of the induction of proliferation or accumulation of regulatoryT cells in the individual.

In one embodiment, administration of a composition described hereincauses induction of the regulatory T cells that are transcription factorFoxp3-positive regulatory T cells or IL-10-producing regulatory T cells.

The composition described herein can be administered by a variety ofroutes and in one embodiment, is administered orally to an individual inneed thereof, such as a patient in need thereof. The composition may beadministered in a number of oral forms, such as in spore-form (in a drypowder or dissolved in a liquid formulation), in enteric capsules, insachets, or in a food matrix, such as yogurt, or a drink.

Also provided is a method to predict a subject's response to treatment(predict whether the subject will or will not respond to treatment) withcompositions of the invention. The method comprises (a) obtaining a (atleast one, one or more) sample, such as a fecal sample or a colonicbiopsy, from a patient before he or she is treated with a bacterialcomposition described herein; (b) measuring or determining thepercentage or absolute counts in the sample of at least one bacterialspecies selected from the group consisting of: Clostridiumsaccharogumia, Clostridium ramosum JCM1298, Clostridium ramosum,Flavonifractor plautii, Pseudoflavonifractor capillosus ATCC 29799,Clostridium hathewayi, Clostridium saccharolyticum WM1, Bacteroides sp.MANG, Clostridium saccharolyticum, Clostridium scindens, Lachnospiraceaebacterium 5_1_57FAA, Lachnospiraceae bacterium 6_1_63FAA, Clostridiumsp. 14616, Clostridium bolteae ATCC BAA-613, cf. Clostridium sp. MLG055,Erysipelotrichaceae bacterium 2_2_44A, Clostridium indolis, Anaerostipescaccae, Clostridium bolteae, Lachnospiraceae bacterium DJF_VP30,Lachnospiraceae bacterium 3_1_57FAA_CT1, Anaerotruncus colihominis,Anaerotruncus colihominis DSM 17241, Ruminococcus sp. ID8,Lachnospiraceae bacterium 2_1_46FAA, Clostridium lavalense, Clostridiumasparagiforme DSM 15981, Clostridium symbiosum, Clostridium symbiosumWAL-14163, Eubacterium contortum, Clostridium sp. D5, Oscillospiraceaebacterium NML 061048, Oscillibacter valericigenes, Lachnospiraceaebacterium A4, Clostridium sp. 316002/08, and Clostridiales bacterium1_7_47FAA, Blautia cocoides, and Anaerostipes caccae DSM 14662, therebyproducing a percentage or count, and (c) comparing the resultingpercentage or count (measurement) to a baseline value of the samemeasurement in a healthy subject, wherein a percentage or count in thesample obtained from the patient that is lower than the baseline valueindicates that the subject may respond favorably to administration ofthe bacterial composition. In some embodiments, the method furthercomprises (d) administering the bacterial composition to the patient ifthe percentage or count in the sample obtained from the patient is lowerthan the baseline value. Optionally, the method may further comprisemeasuring in a patient's sample (e.g., a fecal sample or a colonicbiopsy) the percentages or absolute counts of other commensal speciesthat belong to Clostridium Clusters IV and XIVa, but are not present inthe bacterial composition, wherein a value of the percentage or absolutecount (measurement) lower than baseline further indicates that thesubject may respond favorably to administration of the bacterialcompositions. In some embodiments, the method further comprisesadministering the bacterial composition to the patient if the value ofthe percentage or absolute count (measurement) is lower than baseline.In one embodiment, the patient being assessed suffers from inflammatorybowel disease or a C. difficile infection.

Also provided is a method of monitoring a subject's response totreatment with the bacterial compositions of the invention, comprising:(a) obtaining a (at least one) sample, such as a fecal sample or acolonic biopsy from a patient before treatment with a bacterialcomposition described herein; (b) obtaining, a (at least one)corresponding sample from the patient after treatment with a bacterialcomposition described herein; and (c) comparing the percentage orabsolute counts of at least one bacterial species selected from thegroup consisting of: Clostridium saccharogumia, Clostridium ramosumJCM1298, Clostridium ramosum, Flavonfractor plautii,Pseudoflavonifractor capillosus ATCC 29799, Clostridium hathewayi,Clostridium saccharolyticum WM1, Bacteroides sp. MANG, Clostridiumsaccharolyticum, Clostridium scindens, Lachnospiraceae bacterium5_1_57FAA, Lachnospiraceae bacterium 6_1_63FAA, Clostridium sp. 14616,Clostridium bolteae ATCC BAA-613, cf. Clostridium sp. MLG055,Erysipelotrichaceae bacterium 2_2_44A, Clostridium indolis, Anaerostipescaccae, Clostridium bolteae, Lachnospiraceae bacterium DJF_VP30,Lachnospiraceae bacterium 3_1_57FAA_CT1, Anaerotruncus colihominis,Anaerotruncus colihominis DSM 17241, Ruminococcus sp. ID8,Lachnospiraceae bacterium 2_1_46FAA, Clostridium lavalense, Clostridiumasparagiforme DSM 15981, Clostridium symbiosum, Clostridium symbiosumWAL-14163, Eubacterium contortum, Clostridium sp. D5, Oscillospiraceaebacterium NML 061048, Oscillibacter valericigenes, Lachnospiraceaebacterium A4, Clostridium sp. 316002/08, and Clostridiales bacterium1_7_47FAA, Blautia cocoides, Anaerostipes caccae DSM 14662 in the sampleobtained in (a) with the percentage or absolute counts of the same atleast one bacterial species in the sample obtained in (b), wherein ahigher value in the sample obtained in (b) (after treatment with thebacterial composition) than in the sample obtained in (a) (beforetreatment) indicates that the subject has responded favorably totreatment (e.g. is a positive indicator of enhanced immunosuppression inthe subject). In some embodiments, the method further comprises (d)further administering the bacterial composition to the patient orceasing administration of the bacterial composition to the patient basedon the comparison in (c). Optionally, the method may further comprisemeasuring in the subject's samples the percentages or absolute counts ofother commensal species that belong to Clostridium Clusters IV and XIVa,but are not present in the bacterial composition, wherein a higher valueafter treatment than before treatment indicates that the subject hasresponded favorably to treatment.

Effects of Compositions and Methods Described Herein

The compositions described herein, which contain, as an activecomponent, selected bacteria belonging to the Clostridia class or otherbacteria, as described herein; a culture supernatant of such bacteria; aphysiologically active substance derived from such bacteria; or acombination of two or three of the foregoing are excellent at inducingthe proliferation or accumulation of regulatory T cells (Treg cells).

Immunity in an individual can be suppressed through administration ofthe subject composition, such as through ingestion of the bacterialcomposition in a food or beverage or as a dietary supplement or throughadministration of a pharmaceutical composition comprising the bacterialcomposition. The subject composition can be used, for example, toprevent or treat autoimmune diseases, allergic diseases, infectiousdiseases, as well as to suppress immunological rejection in organtransplantation or the like. In addition, if a food or beverage, such asa health food, comprises the subject composition, healthy individualscan ingest the composition easily and routinely. As a result, it ispossible to induce the proliferation and/or accumulation of regulatory Tcells and thereby to improve immune functions.

The composition described herein provides for a natural, long-lasting,patient-friendly, and benign treatment alternative for immune-mediatedconditions. For example, inflammatory bowel disease is currently managedwith synthetic drugs that may have severe side effects (such ascorticosteroids, TNF inhibitors), cannot be administered orally (such asTNF inhibitors), have inconvenient dosing involving several pills a day(such as mesalazine or sulfasalazine) or have limited efficacy andshort-lived effects (such as currently marketed probiotics, e.g.Lactobacillus GG, Lactobacillus acidophilus, Bifidobacterium longum,etc).

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1A is a histogram showing Foxp3 expression gated CD4 cells fromcolonic lamina propia (C LPL, left panel) and small intestinal laminapropria (SI LPL, right panel) of GF mice or GF mice colonized withuntreated (+huUT, n=4, numbering from #A1 to #A4) or chloroform-treated(+huChloro, n=4, numbering from #B1 to #B4) human feces. Numbers abovebracketed lines indicate the percentage of the population.

FIG. 1B is a histogram showing Helios expression in Foxp3⁺ CD4⁺ cellsfrom colonic lamina propia (left panel) and small intestinal laminapropria (right panel) of GF mice or GF mice colonized with untreated(+huUT) or chloroform-treated (+huChloro) human feces. Numbers abovebracketed lines indicate the percentage of the population.

FIGS. 1C-1D are graphs showing, respectively, combined data for Foxp3expression in CD4+ cells, and for Helios expression in Foxp3⁺ CD4⁺cells, from colonic lamina propia (left panel) and small intestinallamina propria (right panel) of GF mice or GF mice colonized withuntreated (+huUT) or chloroform-treated (+huChloro) human feces. Eachcircle in FIG. 1C and FIG. 1D represents a separate animal, and errorbars indicate the SD. *P<0.05; **P<0.001, unpaired t test.

FIG. 1E shows representative flow cytometry dot plots for theintracellular expressions of IL-17 and IFN− in CD4⁺ cells from coloniclamina propia (upper panel) and small intestinal lamina propria (lowerpanel) of GF mice or GF mice colonized with untreated (+huUT) orchloroform-treated (+huChloro) human feces. The number in each quadrantindicates the percentage of the population.

FIGS. 1F-1G show, respectively, combined data of all mice for IL-17 andIFN-expression in CD4+ cells from colonic lamina propia (left panel) andsmall intestinal lamina propria (right panel) of GF mice or GF micecolonized with untreated (+huUT) or chloroform-treated (+huChloro) humanfeces. Each circle in FIG. 1F and FIG. 1G represents a separate animal,and error bars indicate the SD. *P<0.05; ns, not significant (P>0.05),unpaired t test.

FIGS. 2A-2C shows representative plots (FIG. 2A) and combined data(FIGS. 2B-2C) for Foxp3 expression in CD4+ cells (upper panel in FIG.2A, left panel in FIG. 2B), or Helios expression in Foxp3+CD4+ cells(lower panel in FIG. 2A, right panel in FIG. 2C) for GF mice and GF miceorally inoculated (once a week for 4 weeks) with a suspension ofchloroform-treated human feces that had been previously autoclaved.Numbers above bracketed lines in FIG. 2A indicate the percentage of thepopulation. Each circle in FIG. 2B and FIG. 2C represents a separateanimal, and error bars indicate the SD. ns, not significant (P>0.05),unpaired t test.

FIGS. 3A-3B shows representative plots (FIG. 3A, data of mouse #C4 isshown here) and combined data (FIG. 3B) for Foxp3 expression in CD4+cells from colonic and small intestinal lamina propria lymphocytes forGF mice and GF mice orally inoculated with chloroform-treated humanfeces (+huChloro, n=7, numbering from #C1 to #C7). Numbers abovebracketed lines in FIG. 3A indicate the percentage of the population.Each circle in FIG. 3B represents a separate animal, and error barsindicate the SD. **P<0.001, unpaired t test.

FIGS. 4A-4B shows representative plots (FIG. 4A) and combined data (FIG.4B) for Foxp3 expression in CD4⁺ cells from colonic lamina propria (CLPL) and small intestinal lamina propria (SI LPL) for GF mice and GF(numbering from #D1 to #D6) that were co-housed with #C6 and #C7 ex-GFmice colonized with chloroform-treated human feces. Numbers abovebracketed lines in FIG. 4A indicate the percentage of the population.Each circle in FIG. 4B represents a separate animal, and error barsindicate the SD. **P<0.001, unpaired t test.

FIGS. 5A-5C shows representative plots and combined data for Foxp3expression in CD4+ cells (FIGS. 5A, 5B), or Helios expression in Foxp3⁺CD4⁺ cells (FIG. 5C) from colonic lamina propria (C LPL) and smallintestinal lamina propria (SI LPL) for GF mice, GF mice that wereinoculated with 2000-fold (+x2000, n=4, numbering from #E1 to #E4) or20000-fold (+x20000, n=8, numbering from #F1 to #F8) diluted fecalsuspension from #C4 mouse. Numbers above bracketed lines in FIG. 5Aindicate the percentage of the population. Each circle in FIG. 5B andFIG. 5C represents a separate animal, and error bars indicate the SD.*P<0.05; **P<0.001, unpaired t test.

FIGS. 6A-6D shows representative plots (FIGS. 6A, 6B) and combined data(FIGS. 6C, 6D) for Foxp3 expression in CD4⁺ cells (FIGS. 6A, 6C), orHelios expression in Foxp3⁺ CD4⁺ cells (FIGS. 6B, 6D) from coloniclamina propria (C LPL) and small intestinal lamina propria (SI LPL) forGF mice, and GF mice that were inoculated with fecal suspension of #F3(n=5), #F7 (n=4) or #F8 (n=4) mouse. Numbers above bracketed lines inFIG. 6A and FIG. 6B indicate the percentage of the population. Eachcircle in FIG. 6C and FIG. 6D represents a separate animal, and errorbars indicate the SD. *P<0.05; **P<0.001, unpaired t test.

FIG. 7A-7C shows representative plots (FIG. 7A) and combined data (FIGS.7B, 7C) for Foxp3 expression in CD4+ cells (FIGS. 7A, 7B) or Heliosexpression in Foxp3⁺ CD4⁺ cells for GF mice and GF mice that wereinoculated with 3 isolated strains of bacteria from cecal content of #F8mouse (n=4, numbering from #J1 to #J4). Numbers above bracketed lines inFIG. 7A indicate the percentage of the population. Each circle in FIG.7B and FIG. 7C represents a separate animal, and error bars indicate theSD. ns, not significant (P>0.05), unpaired t test.

FIG. 8 shows the relative abundances of OTUs having the same closestrelative in each cecal sample (bacterial DNA was extracted from thececal contents of mouse #A1, #C4, #F8, #G2, #H3, #I3, #J3 and #K3, shownin the bars). Total number of OTUs detected in each sample is depictedbelow the bar. The detected OTU names in sample #H3, #I3 or #K3, theirclosest relative and their similarity with the closest relative aredepicted in the right table.

FIGS. 9A-9C shows representative plots (FIG. 9A) and combined data(FIGS. 9B, 9C) for Foxp3 expression in CD4⁺ cells (FIGS. 9A, 9B), orHelios expression in Foxp3⁺ CD4⁺ cells (FIGS. 9A, 9C) from coloniclamina propria (C LPL) and small intestinal lamina propria (SI LPL) forGF mice and GF mice that were inoculated with bacteria collections fromculture plate of cecal content of #G2 mouse (n=4, numbering from #K1 to#K4. Numbers above bracketed lines in FIG. 9A indicate the percentage ofthe population. Each circle in FIG. 9B and FIG. 9C represents a separateanimal, and error bars indicate the SD. *P<0.05; **P<0.001, unpaired ttest.

FIGS. 10A-10C shows representative plots (FIG. 10A) and combined data(FIGS. 10B, 10C) for Foxp3 expression in CD4⁺ cells (FIG. 10A, 10B), orHelios expression in Foxp3⁺ CD4⁺ cells (FIG. 10A, 10C) from coloniclamina propria (C LPL) and small intestinal lamina propria (SI LPL) forGF mice and GF mice that were inoculated with a mixture of 23 bacterialstrains that were isolated and shown in Table 2 (23mix). Numbers abovebracketed lines in FIG. 10A indicate the percentage of the population.Each circle in FIG. 10B and FIG. 10C represents a separate animal, anderror bars indicate the SD. *P<0.05; **P<0.001, unpaired t test.

FIG. 11 shows a representative plot of the accumulation of Foxp3⁺ CD4⁺cells in adult GF mice that were inoculated with 2×10⁴ to 2×10⁷-folddiluted caecal samples from +huChlo mice. Experiments were performedmore than twice. Error bars indicate SD. **P<0.01, *P<0.05, ascalculated by Student's t-test.

FIG. 12 shows a representative plot of the accumulation of Foxp3⁺ CD4⁺cells in the colon of adult GF mice that were inoculated with a mixtureof 23 bacterial strains that were isolated and shown in Table 2(23-mix), chloroform-treated human feces (+huChlo) and Faecalibacteriumprausnitzii (+Faecali). Error bars indicate SD. **P<0.01, as calculatedby Student's t-test.

FIG. 13 shows a representative plot of the accumulation of Foxp3⁺ CD4⁺cells in adult GF mice that were the secondary (+2×10⁴-re) and tertiary(+2×10⁴-re-re) recipients of inoculations with the caecal content of+2×10⁴ mice, and adult GF mice inoculated with 2×10⁴-fold diluted caecalsamples from +2×10⁴ mice (+(2×10⁴)²).

FIG. 14 shows the results of 16 s rDNA pyrosequencing the caecalcontents from the defined mice (+hu, +huChlo, +2×10⁴, +2×10⁴-re,(+2×10⁴)², +23-mix) using a 454 sequencer. The relative abundance ofOTUs (%) in the caecal bacterial community in each mouse and the closeststrains in the database and the corresponding isolated strain number forthe indicated OTUs are shown.

FIG. 15 shows a representative plot of the accumulation of Foxp3⁺ CD4⁺cells in the colons of adult IQI, BALB and B6 GF mice on inoculationwith a mixture of 17 bacterial strains that were isolated and shown inTable 4 (17-mix), **P<0.01, as calculated by Student's t-test.

FIG. 16 shows a representative plot of the accumulation of Foxp3⁺ CD4⁺cells in adult IQI GF mice mono-colonized with each of the 17 strainslisted in Table 4 (17-mix).

FIG. 17 shows a representative plot of the accumulation of Foxp3⁺ CD4⁺cells in adult IQI GF mice colonized with 3-mix, 5mix-A, 5-mix-B,5-mix-C or 17-mix as listed in Table 4. Circles indicate individualanimals. Experiments were performed more than twice with similarresults. Error bars indicate SD. **P<0.01, *P<0.05, ns, not significant,as calculated by Student's t-test.

FIG. 18 shows a representative plot of the accumulation of Foxp3⁺ CD4⁺cells in adult SPF mice repeatedly inoculated with 17-mix (SPF+17mix;n=5) or control (SPF+cont; n=6). **P<0.01, as calculated by Student'st-test.

FIG. 19 shows the effects of inoculation with 17-mix on an OVA model ofdiarrhea, as measured by a qualitative diarrhea score. *P<0.05, ascalculated by Student's t-test.

FIG. 20 shows the survival of adult mice inoculated with a mixture of 17bacterial strains listed in Table 4 (17-mix) following exposure totrinitrobenzene sulfonic acid (TNBS), an agent used in experimentalmodels of colitis.

FIG. 21 shows the relative abundance of each of the 17-mix strains inthe human fecal microbiota of ulcerative colitis and healthy subjects.The publically available reads of 15 healthy and 20 ulcerative colitissubjects in the MetaHIT database were aligned to the genome of the 17strains. The mean numbers of mapped reads in healthy and UC groups foreach of the 17 strain genomes are shown. Error bars represent SEM.*P<0.05, as calculated by the Student's t-test.

Table 1 shows the numbers of detected reads and the closest relativesfor each OTU obtained from classification of sequences (3400 reads foreach sample) resulting from 16srRNA coding gene amplification and PCRmetasequencing of bacterial DNA extracted from the cecal contents ofmouse #A1, #C4, #F8, #G2, #H3, #I3, #J3 and #K3 (classification on thebasis of sequence similarity, >97% identity to sequences in nucleic aciddatabases using BLAST)

Table 2 shows, for each of seventeen bacterial strains isolated from thececal contents of mouse #F8, #G2, #I1 and #K3 using BL agar or EG agarplates, the closest relative in known species, the maximum similaritywith the closest relative, its classification in the Clostridiaceaecluster, origin of mouse ID, and culture medium for isolation.

Table 3 shows, for each of 31 bacterial strains isolated from the caecalcontents of mouse #F8, #G2, #I1 and #K3 using BL agar or EG agar plates,the closest relative in known species, the maximum similarity with theclosest relative, the database used for BLAST search, and similaritybetween strains.

Table 4 shows 16S rDNA analysis for each of 31 strains that wereisolated. Bacterial DNA was isolated from each of the 31 strains and the16S rDNA of the isolates was amplified by colony-PCR. Each amplified DNAwas purified, sequenced, and aligned using the ClustalW softwareprogram. Based on the sequence of 16S rDNA for each strain, theirclosest species, % similarity with the closest species, and thesimilarity to other strains are shown. Strains that were included in the23-mix, 17-mix, 5-mixA, 5-mixB, 5-mixC, and 3-mix are marked in theright hand column.

DETAILED DESCRIPTION

<Composition Having Effect of Inducing Proliferation or Accumulation ofRegulatory T Cells>

Described herein is a composition that induces proliferation,accumulation of regulatory T cells or both proliferation andaccumulation of regulatory T cells. The composition comprises, as anactive ingredient, one or more of the following: a (at least one, one ormore) organism selected from the group consisting of: Clostridiumsaccharogumia, Clostridium ramosum JCM1298, Clostridium ramosum,Flavonifractor plautii, Pseudoflavonifractor capillosus ATCC 29799,Clostridium hathewayi, Clostridium saccharolyticum WM1, Bacteroides sp.MANG, Clostridium saccharolyticum, Clostridium scindens, Lachnospiraceaebacterium 5_1_57FAA, Lachnospiraceae bacterium 6_1_63FAA, Clostridiumsp. 14616, Clostridium bolteae ATCC BAA-613, cf. Clostridium sp. MLG055,Erysipelotrichaceae bacterium 2_2_44A, Clostridium indolis, Anaerostipescaccae, Clostridium bolteae, Lachnospiraceae bacterium DJF_VP30,Lachnospiraceae bacterium 3_1_57FAA_CT1, Anaerotruncus colihominis,Anaerotruncus colihominis DSM 17241, Ruminococcus sp. ID8,Lachnospiraceae bacterium 2_1_46FAA, Clostridium lavalense, Clostridiumasparagiforme DSM 15981, Clostridium symbiosum, Clostridium symbiosumWAL-14163, Eubacterium contortum, Clostridium sp. D5, Oscillospiraceaebacterium NML 061048, Oscillibacter valericigenes, Lachnospiraceaebacterium A4, Clostridium sp. 316002/08, and Clostridiales bacterium1_7_47FAA, Blautia cocoides, Anaerostipes caccae DSM 14662, a culturesupernatant of one or more of the bacteria, a component of culturemedium in which a (at least one, one or more) bacterium described hereinhas grown, a physiologically active substance derived from a (at leastone; one or more) bacterium described herein; and a (at least one; oneor more) bacterium containing DNA comprising a nucleotide sequencehaving at least 97% homology to the nucleotide sequence of DNA of any ofthe bacterial species described herein, such as those listed above.Bacteria described herein were isolated from human fecal samples usingthe methods outlined in Examples 19 to 28.

The term “regulatory T cells” refers to T cells that suppress anabnormal or excessive immune response and play a role in immunetolerance. The regulatory T cells are typically transcription factorFoxp3-positive CD4-positive T cells. The regulatory T cells of thepresent invention also include transcription factor Foxp3-negativeregulatory T cells that are IL-10-producing CD4-positive T cells.

The term “induces proliferation or accumulation of regulatory T cells”refers to an effect of inducing the differentiation of immature T cellsinto regulatory T cells, which differentiation leads to theproliferation and/or the accumulation of regulatory T cells. Further,the meaning of “induces proliferation or accumulation of regulatory Tcells” includes in-vivo effects, in vitro effects, and ex vivo effects.All of the following effects are included: an effect of inducing in vivoproliferation or accumulation of regulatory T cells throughadministration or ingestion of the aforementioned bacteria belonging tothe Clostridia class, a culture supernatant of the bacteria orsupernatant component(s), or a physiologically active substance derivedfrom the bacteria; an effect of inducing proliferation or accumulationof cultured regulatory T cells by causing the aforementioned bacteriabelonging to the Clostridia class, a culture supernatant of the bacteriaor supernatant component(s), or a physiologically active substancederived from the bacteria to act on the cultured regulatory T cells; andan effect of inducing proliferation or accumulation of regulatory Tcells which are collected from a living organism and which are intendedto be subsequently introduced into a living organism, such as theorganism from which they were obtained or another organism, by causingthe aforementioned bacteria belonging to the Clostridia class, a culturesupernatant of the bacteria or supernatant component(s), or thephysiologically active substance derived from the bacteria to act on theregulatory T cells. The effect of inducing proliferation or accumulationof regulatory T cells can be evaluated, for example, as follows.Specifically, the aforementioned bacteria belonging to the Clostridiaclass, a culture supernatant of the bacteria or supernatantcomponent(s), or a physiologically active substance derived from thebacteria is orally administered to an experimental animal, such as agerm-free mouse, then CD4-positive cells in the colon are isolated, andthe ratio of regulatory T cells contained in the CD4-positive cells ismeasured by flow cytometry (refer to Example 7).

The regulatory T cells whose proliferation or accumulation is induced bythe composition of the present invention are preferably transcriptionfactor Foxp3-positive regulatory T cells or IL-10-producing regulatory Tcells.

In the present invention, “human-derived bacteria” means bacterialspecies that have been isolated from a fecal sample or from agastrointestinal biopsy obtained from a human individual or whoseancestors were isolated from a fecal sample or from a gastrointestinalbiopsy obtained from a human (e.g., are progeny of bacteria obtainedfrom a fecal sample or a gastrointestinal biopsy). For example, thebacterial species may have been previously isolated from a fecal sampleor from a gastrointestinal biopsy obtained from a human and cultured fora sufficient time to generate progeny. The progeny can then be furthercultured or frozen. The human-derived bacteria are naturally occurringcommensals that populate the gastrointestinal tract of humanindividuals, preferably healthy human individuals.

In the present invention, the term “Clostridia class” (as in“compositions containing bacteria belonging to the Clostridia class”)refers to a class of Gram+, obligate anaerobic bacteria belonging to theFirmicutes phylum that have the ability to form spores. It is importantto note that while currently most bacteria in this class are included inthe Clostridiales order, this categorization is still partly based onold methods and is likely to be redefined in the future based on newadvances in sequencing technologies that are enabling sequencing of thefull genomes of bacteria in this class. Table 2 provides a summary ofthe categorization of 17 abundant species belonging to the Clostridiaclass which have been identified by the inventors as strongTreg-inducers and cultured in vitro. All of these species fall, undercurrent categorization rules, in the Clostridiaceae family, and belongto clusters IV, XIVa, XVI, and XVIII.

The composition of the present invention may include one strain alone(only one strain) of any of the aforementioned bacterial species, buttwo or more strains of the bacteria can be used together. For example,one, two, three, four, five, six, seven, eight, nine, ten, eleven,twelve, thirteen, fourteen, fifteen, sixteen or seventeen of the strainslisted in Table 2 or Table 4, in any combination, can be used togetherto affect regulatory T cells. In some embodiments, the 23, 17, 5, or 3species mixes listed in Table 4 can be used together (and administeredin one or several compositions) to affect regulatory T cells. In someembodiments, the following strains can be combined (the compositioncomprises): strain 1 (OTU136, closest species: Clostridiumsaccharogumia, Clostridium ramosum JCM1298), strain 3 (OTU221, closestspecies: Flavonifractor plautii, Pseudoflavonifractor capillosus ATTC29799), strain 4 (OTU9, closest species: Clostridium hathewayi,Clostridium saccharolyticum WM1), strain 5 (OTU296, closest species:Clostridium scindens, Lachnospiraceae bacterium 5_1_57FAA), strain 6(OTU21, closest species: Blautia coccoides, Lachnospiraceae bacterium6_1_63FAA), strain 7 (OUT 166, closest species: Clostridium sp.,Clostridium bolteae ATCC BAA-613), strain 8 (OTU73, closest species: cf.Clostridium sp. MLG055, Erysipelotrichaceae bacterium 2_2_44A), strain 9(OTU174, closest species: Clostridium indolis, Anaerostipes caccae DSM14662), strain 10 (OTU166, closest species: Clostridium bolteae,Clostridiu bolteae ATCC BAA-613), strain 12 (OTU55, closest species:Lachnospiraceae bacterium DJF_VP30, Lachnospiraceae bacterium3_1_57FAA_CT1), strain 13 (OTU337, closest species: Anaerotruncuscolihominis, Anaerotruncus colihominis DSM 17241), strain 14 (OTU314,closest species: Ruminococcus sp. ID8, Lachnospiraceae bacterium2_1_46FAA), strain 15 (OTU195, closest species: Clostridium lavalense,Clostridium asparagiforme DSM 15981), strain 16 (OTU306, closestspecies: Clostridium symbiosum, Clostridium symbiosum WAL-14163), strain18 (OTU46, closest species: Clostridium ramosum, Clostridium ramosum),strain 21 (OTU87, closest species: Eubacterium contortum, Clostridiumsp. D5), strain 23 (OTU152, closest species: Lachnospiraceae bacteriumDJF_VP30, Lachnospiraceae bacterium 3_1_57FAA_CT1), strain 24 (OTU253,closest species: Oscillospiraceae bacterium NML 061048, Oscillibactervalericigenes), strain 25 (OTU259, closest species: Eubacteriumcontortum, Clostridium sp. D5), strain 26 (OTU281, closest species:Clostridium scindens, Lachnospiraceae bacterium 5_1_57FAA), strain 27(OTU288, closest species: Lachnospiraceae bacterium A4, Lachnospiraceaebacterium 3_1_57FAA_CT1), strain 28 (OTU344, closest species:Clostridium sp. 316002/08, Clostridiales bacterium 1_7_47FAA), andstrain 29 (OTU359, closest species: Lachnospiraceae bacterium A4,Lachnospiraceae bacterium 3_1_57FAA_CT1) as described in Table 4.

In some embodiments, the following strains can be combined (thecomposition comprises: strain 1 (OTU136, closest species: Clostridiumsaccharogumia, Clostridium ramosum JCM1298), strain 3 (OTU221, closestspecies: Flavonifractor plautii, Pseudoflavonifractor capillosus ATTC29799), strain 4 (OTU9, closest species: Clostridium hathewayi,Clostridium saccharolyticum WM1), strain 6 (OTU21, closest species:Blautia coccoides, Lachnospiraceae bacterium 6_1_63FAA), strain 7 (OUT166, closest species: Clostridium sp., Clostridium bolteae ATCCBAA-613), strain 8 (OTU73, closest species: cf. Clostridium sp. MLG055,Erysipelotrichaceae bacterium 2_2_44A), strain 9 (OTU174, closestspecies: Clostridium indolis, Anaerostipes caccae DSM 14662), strain 13(OTU337, closest species: Anaerotruncus colihominis, Anaerotruncuscolihominis DSM 17241), strain 14 (OTU314, closest species: Ruminococcussp. ID8, Lachnospiraceae bacterium 2_1_46FAA), strain 15 (OTU195,closest species: Clostridium lavalense, Clostridium asparagiforme DSM15981), strain 16 (OTU306, closest species: Clostridium symbiosum,Clostridium symbiosum WAL-14163), strain 18 (OTU46, closest species:Clostridium ramosum, Clostridium ramosum), strain 21 (OTU87, closestspecies: Eubacterium contortum, Clostridium sp. D5), strain 26 (OTU281,closest species: Clostridium scindens, Lachnospiraceae bacterium5_1_57FAA), strain 27 (OTU288, closest species: LachnospiraceaebacteriumA4, Lachnospiraceae bacterium 3_1_57FAA_CT1), strain 28(OTU344, closest species: Clostridium sp. 316002/08, Clostridialesbacterium 1_7_47FAA), and strain 29 (OTU359, closest species:Lachnospiraceae bacteriumA4, Lachnospiraceae bacterium 3_1_57FAA_CT1) asdescribed in Table 4.

In some embodiments, the following strains can be combined (thecomposition comprises): strain 1 (OTU136, closest species: Clostridiumsaccharogumia, Clostridium ramosum JCM1298), strain 4 (OTU9, closestspecies: Clostridium hathewayi, Clostridium saccharolyticum WM1), strain16 (OTU306, closest species: Clostridium symbiosum, Clostridiumsymbiosum WAL-14163), strain 27 (OTU288, closest species:Lachnospiraceae bacteriumA4, Lachnospiraceae bacterium 3_1_57FAA_CT1),and strain 29 (OTU359, closest species: Lachnospiraceae bacteriumA4,Lachnospiraceae bacterium 3_1_57FAA_CT1) as described in Table 4. Insome embodiments, the following strains can be combined: strain 6(OTU21, closest species: Blautia coccoides, Lachnospiraceae bacterium6_1_63FAA), strain 8 (OTU73, closest species: cf. Clostridium sp.MLG055, Erysipelotrichaceae bacterium 2_2_44A), strain 13 (OTU337,closest species: Anaerotruncus colihominis, Anaerotruncus colihominisDSM 17241), strain 14 (OTU314, closest species: Ruminococcus sp. ID8,Lachnospiraceae bacterium 2_1_46FAA), and strain 26 (OTU281, closestspecies: Clostridium scindens, Lachnospiraceae bacterium 5_1_57FAA) asdescribed in Table 4. In some embodiments, the following strains can becombined: strain 3 (OTU221, closest species: Flavonifractor plautii,Pseudoflavonifractor capillosus ATTC 29799), strain 7 (OUT 166, closestspecies: Clostridium sp., Clostridium bolteae ATCC BAA-613), strain 9(OTU174, closest species: Clostridium indolis, Anaerostipes caccae DSM14662), strain 15 (OTU195, closest species: Clostridium lavalense,Clostridium asparagiforme DSM 15981), and strain 28 (OTU344, closestspecies: Clostridium sp. 316002/08, Clostridiales bacterium 1_7_47FAA)as described in Table 4 In some embodiments, the following strains canbe combined: strain 1 (OTU136, closest species: Clostridiumsaccharogumia, Clostridium ramosum JCM1298), strain 2 (OTU46, closestspecies: Flavonifractor plautii, Pseudoflavonifractor capillosus ATCC29799) and strain 3 (OTU221, closest species: Flavonifractor plautii,Pseudoflavonifractor capillosus ATTC 29799) as described in Table 4. Theuse of multiple strains of the aforementioned species of bacteria,preferably belonging to the Clostridium cluster XIVa or the cluster IVin combination can bring about an excellent effect on regulatory Tcells. In addition to the bacteria belonging to clusters XIVa and IV,Clostridium ramosum, Clostridium saccharogumia (belonging to clusterXVIII) and cf. Clostridium sp. MLG055 (belonging to cluster XVI) canalso be used. If more than one strain of bacteria is used (e.g., one ormore strain belonging to cluster XIVa, one or more strain belonging tocluster IV, one or more strain belonging to clusters XVIII or XVI or acombination of any of the foregoing), the number and ratio of strainsused can vary widely. The number and ratio to be used can be determinedbased on a variety of factors (e.g., the desired effect, such asinduction or inhibition of proliferation or accumulation of regulatory Tcells; the disease or condition to be treated, prevented or reduced inseverity; the age or gender of the recipient; the typical amounts of thestrains in healthy humans). The strains can be present in a singlecomposition, in which case they can be consumed or ingested together (ina single composition), or can be present in more than one composition(e.g., each can be in a separate composition), in which case they can beconsumed individually or the compositions can be combined and theresulting combination (combined compositions) consumed or ingested. Anynumber or combination of the strains that proves effective (e.g., anynumber from one to 22, such as 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to3, 1 to 2, and any number therebetween or one to 23, such as 1 to 23, 3to 23, 5 to 23, 1 to 20, 1 to 17, 3 to 17, 5 to 17, 1 to 15, 1 to 10, 1to 5, 3 to 5, 1 to 3, 1 to 2, and any number therebetween) can beadministered. In certain embodiments of the present invention, acombination of some or all of the 22 or 23 (e.g., the 23 strains inExample 32 and Table 4) strains described in the present disclosure isused. For example, at least one, two or more, three, three or more,four, four or more, five, five or more, six, six or more or any othernumber of the 22 or 23 described strains, including 22 or 23 strains,can be used. In some embodiments, the specific combinations of 3, 5, 17,or 23 strains described in Table 4 can be used (the compositioncomprises combinations of 3, 5, 17 or 23 strains described in Table 4).They can be used in combination with one another and in combination withstrains not described in the cited reference.

Cells of bacteria belonging to the Clostridia class, such as thesespecifically described herein, can be used in spore form or invegetative form. From the viewpoint of stability to high temperature andpressure conditions, extended shelf life, ease of handling, resistanceto antibiotics, and lack of need for a cold chain storage anddistribution, the bacteria may be preferably in the form of spore. Fromthe viewpoint of abiding by the directives of certain manufacturingorganizations that do not tolerate spore contamination in theirfacilities, the bacteria may alternatively be produced (and lateradministered) in the form of vegetative cells.

The term the “physiologically active substance derived from bacteriabelonging to the Clostridia class” of the present invention includessubstances contained in the bacteria, secretion products of thebacteria, and metabolites of the bacteria. Such a physiologically activesubstance can be identified by purifying an active component from thebacteria, a culture supernatant thereof, or intestinal tract contents inthe intestinal tract of a mouse in which only bacteria belonging to theClostridia class are colonized by an already known purification method.

“Chloroform treatment” of a fecal sample obtained from a human is amethod that isolates the bacteria in the fecal sample that have theability to form spores, and is not particularly limited, as long as thespore-forming fraction is obtained by treating feces of a human withchloroform (for example, 3% chloroform), and has the effect of inducingproliferation or accumulation of regulatory T cells, including mammalianregulatory T cells such as murine regulatory T cells and humanregulatory T cells.

When the aforementioned “bacteria belonging to the Clostridia class” arecultured in a medium, substances contained in the bacteria, secretionproducts and metabolites produced by the bacteria are released from thebacteria. The meaning of the active ingredient “culture supernatant ofthe bacteria” in the composition of the present invention includes suchsubstances, secretion products, and metabolites. The culture supernatantis not particularly limited, as long as the culture supernatant has theeffect of inducing proliferation or accumulation of regulatory T cells.Examples of the culture supernatant include a protein fraction of theculture supernatant, a polysaccharide fraction of the culturesupernatant, a lipid fraction of the culture supernatant, and alow-molecular weight metabolite fraction of the culture supernatant.

The bacterial composition may be administered in the form of apharmaceutical composition, a dietary supplement, or a food or beverage(which may also be an animal feed), or may be used as a reagent for ananimal model experiment. The pharmaceutical composition, the dietarysupplement, the food or beverage, and the reagent induce proliferationor accumulation of regulatory T cells. An example presented hereinrevealed that regulatory T cells (Treg cells) induced by bacteria or thelike belonging to the Clostridia class suppressed the proliferation ofeffector T-cells. The composition of the present invention can be usedsuitably as a composition having an immunosuppressive effect. Theimmunosuppressive effect can be evaluated, for example, as follows.Regulatory T cells isolated from an experimental animal, such as amouse, to which the composition of the present invention is orallyadministered are caused to act on effector T-cells (CD4⁺ CD25⁻ cells)isolated from the spleen, and the proliferation ability thereof ismeasured by using the intake amount of [³H]-thymidine as an index (referto Example 14).

The bacterial composition of the present invention can be used, forexample, as a pharmaceutical composition for preventing or treating(reducing, partially or completely, the adverse effects of) anautoimmune disease. such as chronic inflammatory bowel disease, systemiclupus erythematosus, rheumatoid arthritis, multiple sclerosis, orHashimoto's disease; an allergic disease, such as a food allergy,pollenosis, or asthma; an infectious disease, such as an infection withClostridium difficile; an inflammatory disease such as a TNF-mediatedinflammatory disease (e.g., an inflammatory disease of thegastrointestinal tract, such as pouchitis, a cardiovascular inflammatorycondition, such as atherosclerosis, or an inflammatory lung disease,such as chronic obstructive pulmonary disease); a pharmaceuticalcomposition for suppressing rejection in organ transplantation or othersituations in which tissue rejection might occur; a supplement, food, orbeverage for improving immune functions; or a reagent for suppressingthe proliferation or function of effector T-cells.

More specific examples of target diseases for which the composition isuseful for treatment (reducing adverse effects or prevention) includeautoimmune diseases, allergic diseases, infectious diseases, andrejection in organ transplantations, such as inflammatory bowel disease(IBD), ulcerative colitis, Crohn's disease, sprue, autoimmune arthritis,rheumatoid arthritis, Type I diabetes, multiple sclerosis, graft vs.host disease following bone marrow transplantation, osteoarthritis,juvenile chronic arthritis, Lyme arthritis, psoriatic arthritis,reactive arthritis, spondy loarthropathy, systemic lupus erythematosus,insulin dependent diabetes mellitus, thyroiditis, asthma, psoriasis,dermatitis scleroderma, atopic dermatitis, graft versus host disease,acute or chronic immune disease associated with organ transplantation,sarcoidosis, atherosclerosis, disseminated intravascular coagulation,Kawasaki's disease, Grave's disease, nephrotic syndrome, chronic fatiguesyndrome, Wegener's granulomatosis, Henoch-Schoenlejn purpurea,microscopic vasculitis of the kidneys, chronic active hepatitis,uveitis, septic shock, toxic shock syndrome, sepsis syndrome, cachexia,acquired immunodeficiency syndrome, acute transverse myelitis,Huntington's chorea, Parkinson's disease, Alzheimer's disease, stroke,primary biliary cirrhosis, hemolytic anemia, polyglandular deficiencytype I syndrome and polyglandular deficiency type II syndrome, Schmidt'ssyndrome, adult (acute) respiratory distress syndrome, alopecia,alopecia areata, seronegative arthopathy, arthropathy, Reiter's disease,psoriatic arthropathy, chlamydia, yersinia and salmonella associatedarthropathy, spondyloarhopathy, atheromatous disease/arteriosclerosis,allergic colitis, atopic allergy, food allergies such as peanut allergy,tree nut allergy, egg allergy, milk allergy, soy allergy, wheat allergy,seafood allergy, shellfish allergy, or sesame seed allergy, autoimmunebullous disease, pemphigus vulgaris, pemphigus foliaceus, pemphigoid,linear IgA disease, autoimmune haemolytic anaemia, Coombs positivehaemolytic anaemia, acquired pernicious anaemia, juvenile perniciousanaemia, myalgic encephalitis/Royal Free Disease, chronic mucocutaneouscandidiasis, giant cell arteritis, primary sclerosing hepatitis,cryptogenic autoimmune hepatitis, Acquired Immunodeficiency DiseaseSyndrome, Acquired Immunodeficiency Related Diseases, Hepatitis C,common varied immunodeficiency (common variable hypogammaglobulinaemia),dilated cardiomyopathy, fibrotic lung disease, cryptogenic fibrosingalveolitis, postinflammatory interstitial lung disease, interstitialpneumonitis, connective tissue disease associated interstitial lungdisease, mixed connective tissue disease associated lung disease,systemic sclerosis associated interstitial lung disease, rheumatoidarthritis associated interstitial lung disease, systemic lupuserythematosus associated lung disease, dermatomyositis/polymyositisassociated lung disease, Sjogren's disease associated lung disease,ankylosing spondy litis associated lung disease, vasculitic diffuse lungdisease, haemosiderosis associated lung disease, drug-inducedinterstitial lung disease, radiation fibrosis, bronchiolitis obliterans,chronic eosinophilic pneumonia, lymphocytic infiltrative lung disease,postinfectious interstitial lung disease, gouty arthritis, autoimmunehepatitis, type-1 autoimmune hepatitis (classical autoimmune or lupoidhepatitis), type-2 autoimmune hepatitis (anti-LKM antibody hepatitis),autoimmune mediated hypoglycemia, type B insulin resistance withacanthosis nigricans, hypoparathyroidism, acute immune diseaseassociated with organ transplantation, chronic immune disease associatedwith organ transplantation, osteoarthrosis, primary sclerosingcholangitis, idiopathic leucopenia, autoimmune neutropenia, renaldisease NOS, glomerulonephritides, microscopic vasulitis of the kidneys,discoid lupus, erythematosus, male infertility idiopathic or NOS, spermautoimmunity, multiple sclerosis (all subtypes), insulin dependentdiabetes mellitus, sympathetic ophthalmia, pulmonary hypertensionsecondary to connective tissue disease, Goodpasture's syndrome,pulmonary manifestation of polyarteritis nodosa, acute rheumatio fever,rheumatoid spondylitis, Still's disease, systemic sclerosis, Takayasu'sdisease/arteritis, autoimmune thrombocytopenia, idiopathicthrombocytopenia, autoimmune thyroid disease, hyperthyroidism, goitrousautoimmune hypothyroidism (Hashimoto's disease), atrophic autoimmunehypothyroidism, primary myxoedema, phacogenic uveitis, primaryvasculitis, vitiligo, allergic rhinitis (pollen allergies), anaphylaxis,pet allergies, latex allergies, drug allergies, allergicrhinoconjuctivitis, eosinophilic esophagitis, hypereosinophilicsyndrome, eosinophilic gastroenteritis cutaneous lupus erythematosus,eosinophilic esophagitis, hypereosinophilic syndrome, and eosinophilicgastroenteritis, and diarrhea.

Additional examples of target diseases for which the composition isuseful for treatment include colon cancer, cystic fibrosis, celiacdisease, Type 2 diabetes, and autism-related immunopathologies. Thesediseases are characterized by a reduction of Clostridium Clusters IV andXIV in the gastrointestinal microbiota.

Compositions described herein can also be used as a pharmaceuticalcomposition for preventing or treating infectious diseases in anindividual whose resistance to the infectious diseases is impaired, forexample because of damage due to excessive inflammation caused by theimmunity or due to an alteration of the patient's microbiome. Examplesof infectious pathogens that impair maintenance or recovery ofhomeostasis of a host, and which eventually bring about suchimmunopathological tissue damage include Salmonella, Shigella,Clostridium difficile, Mycobacterium (which cause the diseasetuberculosis), protozoa (which cause malaria), filarial nematodes (whichcause the disease filariasis), Schistosoma (which causeschistosomiasis), Toxoplasma (which cause the disease toxoplasmosis),Leishmania (which cause the disease leishmaniasis), HCV and HBV (whichcause the disease hepatitis C and hepatitis B), and herpes simplexviruses (which cause the disease herpes).

Pharmaceutical preparations can be formulated from the bacterialcompositions described by drug formulation methods known to those ofskill in the art. For example, the composition can be used orally orparenterally in the form of capsules, tablets, pills, sachets, liquids,powders, granules, fine granules, film-coated preparations, pellets,troches, sublingual preparations, chewables, buccal preparations,pastes, syrups, suspensions, elixirs, emulsions, liniments, ointments,plasters, cataplasms, transdermal absorption systems, lotions,inhalations, aerosols, injections, suppositories, and the like.

For formulating these preparations, the bacterial compositions can beused in appropriate combination with carriers that are pharmacologicallyacceptable or acceptable for ingestion, such as in a food or beverage,including one or more of the following: sterile water, physiologicalsaline, vegetable oil, solvent, a base material, an emulsifier, asuspending agent, a surfactant, a stabilizer, a flavoring agent, anaromatic, an excipient, a vehicle, a preservative, a binder, a diluent,a tonicity adjusting agent, a soothing agent, a bulking agent, adisintegrating agent, a buffer agent, a coating agent, a lubricant, acolorant, a sweetener, a thickening agent, a flavor corrigent, asolubilizer, and other additives.

A pharmaceutical preparation or formulation and particularly apharmaceutical preparation for oral administration, comprises anadditional component that enables efficient delivery of the bacterialcomposition of the present invention to the colon, in order to moreefficiently induce proliferation or accumulation of regulatory T cellsin the colon. A variety of pharmaceutical preparations that enable thedelivery of the bacterial composition to the colon can be used. Examplesthereof include pH sensitive compositions, more specifically, bufferedsachet formulations or enteric polymers that release their contents whenthe pH becomes alkaline after the enteric polymers pass through thestomach. When a pH sensitive composition is used for formulating thepharmaceutical preparation, the pH sensitive composition is preferably apolymer whose pH threshold of the decomposition of the composition isbetween about 6.8 and about 7.5. Such a numeric value range is a rangein which the pH shifts toward the alkaline side at a distal portion ofthe stomach, and hence is a suitable range for use in the delivery tothe colon.

Another embodiment of a pharmaceutical preparation useful for deliveryof the bacterial composition to the colon is one that ensures thedelivery to the colon by delaying the release of the contents (e.g., thebacterial composition) by approximately 3 to 5 hours, which correspondsto the small intestinal transit time. In one embodiment of apharmaceutical preparation for delayed release, a hydrogel is used as ashell. The hydrogel is hydrated and swells upon contact withgastrointestinal fluid, with the result that the contents areeffectively released (released predominantly in the colon). Delayedrelease dosage units include drug-containing compositions having amaterial which coats or selectively coats a drug or active ingredient tobe administered. Examples of such a selective coating material includein vivo degradable polymers, gradually hydrolyzable polymers, graduallywater-soluble polymers, and/or enzyme degradable polymers. A widevariety of coating materials for efficiently delaying the release isavailable and includes, for example, cellulose-based polymers such ashydroxypropyl cellulose, acrylic acid polymers and copolymers such asmethacrylic acid polymers and copolymers, and vinyl polymers andcopolymers such as polyvinylpyrrolidone.

Examples of the composition enabling the delivery to the colon furtherinclude bioadhesive compositions which specifically adhere to thecolonic mucosal membrane (for example, a polymer described in thespecification of U.S. Pat. No. 6,368,586) and compositions into which aprotease inhibitor is incorporated for protecting particularly abiopharmaceutical preparation in the gastrointestinal tracts fromdecomposition due to an activity of a protease.

An example of a system enabling the delivery to the colon is a system ofdelivering a composition to the colon by pressure change in such a waythat the contents are released by utilizing pressure change caused bygeneration of gas in bacterial fermentation at a distal portion of thestomach. Such a system is not particularly limited, and a more specificexample thereof is a capsule which has contents dispersed in asuppository base and which is coated with a hydrophobic polymer (forexample, ethyl cellulose).

Another example of the system enabling the delivery to the colon is asystem of delivering a composition to the colon, the system beingspecifically decomposed by an enzyme (for example, a carbohydratehydrolase or a carbohydrate reductase) present in the colon. Such asystem is not particularly limited, and more specific examples thereofinclude systems which use food components such as non-starchpolysaccharides, amylose, xanthan gum, and azopolymers.

When used as a pharmaceutical preparation, the bacterial composition maybe used in combination with an already known pharmaceutical compositionfor use in immunosuppression. In some embodiments, the pharmaceuticalpreparation can comprise both the bacterial composition and the alreadyknown pharmaceutical composition. Such a known pharmaceuticalcomposition is not particularly limited, and may be at least onetherapeutic composition selected from the group consisting ofcorticosteroids, mesalazine, mesalamine, sulfasalazine, sulfasalazinederivatives, immunosuppressive drugs, cyclosporin A, mercaptopurine,azathiopurine, prednisone, methotrexate, antihistamines,glucocorticoids, epinephrine, theophylline, cromolyn sodium,anti-leukotrienes, anti-cholinergic drugs for rhinitis, anti-cholinergicdecongestants, mast-cell stabilizers, monoclonal anti-IgE antibodies,vaccines (preferably vaccines used for vaccination where the amount ofan allergen is gradually increased), anti-TNF inhibitors such asinfliximab, adalimumab, certolizumab pegol, golimumab, or etanercept,and combinations thereof. It is preferable to use these therapeuticcompositions in combination with the bacterial composition describedherein. The bacterial composition can also be used as an adjuvant toimprove the efficacy of a vaccine formulation such as a vaccine for theprophylaxis or treatment of an autoimmune disease or an allergicdisease.

The bacterial composition can be used as a food or beverage, such as ahealth food or beverage, a food or beverage for infants, a food orbeverage for pregnant women, athletes, senior citizens or otherspecified group, a functional food, a beverage, a food or beverage forspecified health use, a dietary supplement, a food or beverage forpatients, or an animal feed. Specific examples of the foods andbeverages include various beverages such as juices, refreshingbeverages, tea beverages, drink preparations, jelly beverages, andfunctional beverages; alcoholic beverages such as beers;carbohydrate-containing foods such as rice food products, noodles,breads, and pastas; paste products such as fish hams, sausages, pasteproducts of seafood; retort pouch products such as curries, food dressedwith a thick starchy sauces, and Chinese soups; soups; dairy productssuch as milk, dairy beverages, ice creams, cheeses, and yogurts;fermented products such as fermented soybean pastes, yogurts, fermentedbeverages, and pickles; bean products; various confectionery productssuch as Western confectionery products including biscuits, cookies, andthe like, Japanese confectionery products including steamed bean-jambuns, soft adzuki-bean jellies, and the like, candies, chewing gums,gummies, cold desserts including jellies, crème caramels, and frozendesserts; instant foods such as instant soups and instant soy-beansoups; microwavable foods; and the like. Further, the examples alsoinclude health foods and beverages prepared in the forms of powders,granules, tablets, capsules, liquids, pastes, and jellies. Thecomposition of the present invention can be used for animals, includinghumans. The animals, other than humans, are not particularly limited,and the composition can be used for various livestock, poultry, pets,experimental animals, and the like. Specific examples of the animalsinclude pigs, cattle, horses, sheep, goats, chickens, wild ducks,ostriches, domestic ducks, dogs, cats, rabbits, hamsters, mice, rats,monkeys, and the like, but the animals are not limited thereto.

Without wishing to be bound by theory, individuals in whom bacteriabelonging to the group Firmicutes (the group to which the Clostridiumclusters IV and XIVa belong) are relatively abundant gain more bodyweight than individuals in whom bacteria belonging to the groupBacteroidetes are relatively abundant is large. The bacterialcomposition is capable of conditioning absorption of nutrients andimproving feed efficiency. From such a viewpoint, the bacterialcomposition can be used for promoting body weight gain, or for a highefficiency animal feed. Diseases and conditions that would benefit frombody weight gain include, e.g., starvation, cancer, AIDS,gastrointestinal disorders (e.g., celiac disease, peptic ulcer,inflammatory bowel disease (Crohns' disease and ulcerative colitis),pancreatitis, gastritis, diarrhea), hyperthyroidism, infection, renaldisease, cardiac disease, pulmonary disease, connective tissue disease,weight loss caused by medications, anorexia, Addison's disease,dementia, depression, hypercalcemia, Parkinson's disease andtuberculosis.

The addition of the bacterial composition to an antibiotic-free animalfeed makes it possible to increase the body weight of an animal thatingests the animal feed to a level equal to or higher than that achievedby animal ingesting antibiotic-containing animal feeds, and also makesit possible to reduce pathogenic bacteria in the stomach to a levelequal to those in animals consuming typical antibiotic-containing animalfeeds. The bacterial composition can be used as a component of an animalfeed that does not need the addition of antibiotics.

In addition, unlike conventional bacteria (Lactobacillus andBifidobacteria) in commercial use, which are not easy to incorporateinto the livestock production, the present bacterial composition inspore form can be pelletized, sprayed, or easily mixed with an animalfeed and can also be added to drinking water.

Animal feed comprising the bacterial composition can be fed to a widevariety of types of animals and animals of a varying ages and can be fedat regular intervals or for a certain period (for example, at birth,during weaning, or when the animal is relocated or shipped).

The bacterial composition can be used to promote weight gain and enhanceenergy absorption in humans and nonhumans (e.g., farm or other foodanimals).

The bacterial active components of the bacterial composition can bemanufactured using fermentation techniques well known in the art. In oneembodiment, the active ingredients are manufactured using anaerobicfermentors, which can support the rapid growth of bacterial speciesbelonging to the Clostridia class. The anaerobic fermentors may be, forexample, stirred tank reactors or disposable wave bioreactors. Culturemedia such as BL media and EG media, or similar versions of these mediadevoid of animal components can be used to support the growth of thebacterial species. The bacterial product can be purified andconcentrated from the fermentation broth by traditional techniques, suchas centrifugation and filtration, and can optionally be dried andlyophilized by techniques well known in the art.

A food or beverage comprising a bacterial composition described hereincan be manufactured by manufacturing techniques well known in thetechnical field. One or more components (for example, a nutrient) whichare effective for the improvement of an immune function by animmunosuppressive effect may be added to the food or beverage. Inaddition, the food or beverage may be combined with another component oranother functional food exhibiting a function other than the function ofthe improvement of an immune function to thereby serve as amulti-functional food or beverage.

Moreover, the bacterial composition can be incorporated into foodsrequiring a processing step which may destroy ordinary probioticstrains. Specifically, most commercially usable probiotic strains cannotbe incorporated into foods that need to be processed, for example, byheat treatment, long term storage, freezing, mechanical stress, orhigh-pressure treatment (for example, extrusion forming or rollforming). On the other hand, because of the advantageous nature offorming spores, the bacterial composition described herein can be easilyincorporated into such processed foods. For example, the bacterialcomposition in the form of spores can survive even in a dried food, andcan remain living even after being ingested. The bacterial compositioncan withstand low-temperature sterilization processes, typicallyprocesses carried out at a temperature from about 70° C. to about 100°C., both inclusive. The bacterial composition can be incorporated intodairy products that require a pasteurization step. Furthermore, thebacterial composition can withstand long-term storage of many years;high-temperature processing such as baking and boiling; low-temperatureprocessing such as freezing and cold storage; and high-pressuretreatments such as extrusion forming and roll forming.

Many types of foods that need to be processed under such harshconditions include foods which need to be processed in a microwave ovento be edible (for example, oatmeal), foods which need to be baked to beedible (for example, a muffin), foods which need to be subjected to asterilization high-temperature treatment for a short period of time tobe edible (for example, milk), and foods which need to be heated to bedrinkable (for example, hot tea).

The amount of the bacterial composition to be administered or ingestedcan be determined empirically, taking into consideration such factors asthe age, body weight, gender, symptoms, health conditions, of anindividual who will receive it, as well as the kind of bacterialcomposition (a pharmaceutical product, a food or beverage) to beadministered or ingested. For example, the amount per administration oringestion is generally 0.01 mg/kg body weight to 100 mg/kg body weight,and, in specific embodiments, 1 mg/kg body weight to 10 mg/kg bodyweight. Also described herein is a method for suppressing the immunity(reducing the immune response) of a subject, the method beingcharacterized in that the bacteria belonging to the Clostridia class orthe physiologically active substance derived from the bacteria isadministered to or ingested by the subject as described above.

The bacterial composition may be administered to an individual once, orit may be administered more than once. If the composition isadministered more than once, it can be administered on a regular basis(for example, once a day, once every two days, once a week, once everytwo weeks, once a month, once every 6 months, or once a year) or on anas needed or irregular basis. The appropriate frequency ofadministration (which may depend on host genetics, age, gender, andhealth or disease status of the subject, among other factors) may bedetermined empirically. For example, a patient can be administered onedose of the composition, and the levels of the bacterial strains of thecomposition in fecal samples obtained from the patient can be measuredat different times (for example after 1 day, after 2 days, after 1 week,after 2 weeks, after 1 month). When the levels of the bacteria fall to,for example, one half of their maximum value, a second dose can beadministered, and so on.

A product comprising the bacterial composition (a pharmaceuticalproduct, a food or beverage, or a reagent) or a manual thereof may beaccompanied by document or statement explaining that the product can beused to suppress the immunity (including a statement that the producthas an immunosuppressive effect and a statement that the product has aneffect of suppressing the proliferation or function of effectorT-cells). Here, the “provision to the product or the manual thereof withthe note” means that the document or statement is provided to a mainbody, a container, a package, or the like of the product, or the note isprovided to a manual, a package insert, a leaflet, or other printedmatters, which disclose information on the product.

<Method for Inducing Proliferation or Accumulation of Regulatory TCells>

As described above, and as shown in Examples, administration of thebacterial composition to an individual makes it possible to induceproliferation or accumulation of regulatory T cells in the individual.This provides a method of inducing proliferation or accumulation ofregulatory T cells in an individual, the method comprising:administering, to the individual, at least one substance selected fromthe group consisting of: (a) Clostridium saccharogumia, Clostridiumramosum JCM1298, Clostridium ramosum, Flavonifractor plautii,Pseudoflavonifractor capillosus ATCC 29799, Clostridium hathewayi,Clostridium saccharolyticum WM1, Bacteroides sp. MANG, Clostridiumsaccharolyticum, Clostridium scindens, Lachnospiraceae bacterium5_1_57FAA, Lachnospiraceae bacterium 6_1_63FAA, Clostridium sp. 14616,Clostridium bolteae ATCC BAA-613, cf. Clostridium sp. MLG055,Erysipelotrichaceae bacterium 2_2_44A, Clostridium indolis, Anaerostipescaccae, Clostridium bolteae, Lachnospiraceae bacterium DJF_VP30,Lachnospiraceae bacterium 3_1_57FAA_CT1, Anaerotruncus colihominis,Anaerotruncus colihominis DSM 17241, Ruminococcus sp. ID8,Lachnospiraceae bacterium 2_1_46FAA, Clostridium lavalense, Clostridiumasparagiforme DSM 15981, Clostridium symbiosum, Clostridium symbiosumWAL-14163, Eubacterium contortum, Clostridium sp. D5, Oscillospiraceaebacterium NML 061048, Oscillibacter valericigenes, Lachnospiraceaebacterium A4, Clostridium sp. 316002/08, and Clostridiales bacterium1_7_47FAA, Blautia cocoides, Anaerostipes caccae DSM 14662; (b) aculture supernatant of at least one (a, one or more) of the bacteriadescribed/listed herein; (c) a physiologically active substance derivedfrom a (one or more, at least one) bacterium described/listed herein; ora combination of any two or three of (a), (b) and (c). The bacterialcomposition is administered (provided) to the individual in sufficientquantity to produce the desired effect of inducing proliferation,accumulation or both proliferation and accumulation of regulatory Tcells. It may be administered to an individual in need of treatment,reduction in the severity of or prevention of at least one diseaseselected from an autoimmune disease, an inflammatory disease, anallergic disease, and an infectious disease.

Note that, the “individual” or “subject” may be in a healthy state or adiseased state. The method may further comprise the optional step ofadministering at least one (a, one or more) antibiotic preceding, or incombination with, the bacterial composition. The antibiotic administeredcan be, for example, one which facilitates recolonization of the gut byGram-positive bacteria of the Clostridia class, such as an antibioticthat reduces Gram-negative bacteria. Examples of such antibioticsinclude aminoglycoside antibiotics (amikacin, gentamicin, kanamycin,neomycin, netilmicin, tobramycin, and paromomycin), cephalosporinantibiotics (cefaclor, cefamandole, cefoxitin, cefprozil, cefuroxime,cefixime, cefdinir, cefditoren, cefoperazone, cefotaxime, ceftazidime,ceftibuten, ceftizoxime, ceftriaxone, and cefoxotin), sulfonamides,ampicillin, and streptomycin.

Moreover, a prebiotic composition such as almond skin, inulin,oligofructose, raffinose, lactulose, pectin, hemicellulose (such asxyloglucan and alpha-glucans), amylopectin, and resistant starch whichare not decomposed in the upper gastrointestinal tract and promote thegrowth of intestinal microbes in the intestinal tract, as well as growthfactors such as acetyl-Co A, biotin, beet molasses, and yeast extracts,preferentially contributes to the proliferation of the bacterial speciesin the composition belonging to the Clostridia class. A method ofinducing proliferation and/or accumulation of regulatory T cells in anindividual can comprise administering, to the individual, at least onesubstance selected from the above in combination with the bacterialcomposition. Also contemplated herein is a composition comprising thebacterial composition and a prebiotic composition.

The above-described antibiotic, and the above-described prebioticcomposition or growth factor may be used in combination. Moreover, atherapeutic composition may be administered to an individual togetherwith at least one substance selected from the group consisting of thebacterial composition, an antibiotic, and a prebiotic composition orgrowth factor.

A therapeutic composition can be, for example, one therapeuticcomposition selected from the group consisting of corticosteroids,mesalazine, mesalamine, sulfasalazine, sulfasalazine derivatives,immunosuppressive drugs, cyclosporin A, mercaptopurine, azathiopurine,prednisone, methotrexate, antihistamines, glucocorticoids, epinephrine,theophylline, cromolyn sodium, anti-leukotrienes, anti-cholinergic drugsfor rhinitis, anti-cholinergic decongestants, mast-cell stabilizers,monoclonal anti-IgE antibodies, vaccines (preferably, vaccines used forvaccination where the amount of an allergen is gradually increased),anti-TNF inhibitors such as infliximab, adalimumab, certolizumab pegol,golimumab, or etanercept, and combinations thereof. These therapeuticcompositions can be administered prior to, in combination with orfollowing administration of the bacterial composition and optionally,also in combination with an antibiotic, a prebiotic composition, agrowth factor or any combination of an antibiotic, a prebioticcomposition and a growth factor.

There is no particular limitation imposed on the combined use of thetherapeutic composition with at least one substance selected from thegroup consisting of the bacterial composition, the “antibiotic”, and the“prebiotic composition or growth factor”. For example, the “onesubstance” and the therapeutic composition are administered orally orparenterally to an individual simultaneously orsequentially/individually at any appropriate time.

Whether administration of the bacterial composition induces theproliferation and/or accumulation of regulatory T cells can bedetermined by using, as an index, increase or reinforcement of at leastone of the following: the number of regulatory T cells, the ratio ofregulatory T cells in the T cell group of the colon, a function ofregulatory T cells, or expression of a marker of regulatory T cells. Aspecific approach is measurement counts or percentage ofFoxp3-expressing Tregs in a patient sample, such as a biopsy or a bloodsample, promotion (enhancement) of IL-10 expression, promotion(enhancement) of CTLA4 expression, promotion (enhancement) of IDOexpression, suppression of IL-4 expression, or colonization of anindividual with the bacterial composition administered as the index ofthe induction of proliferation or accumulation of regulatory T cells.

Methods for detecting such expression include northern blotting, RT-PCR,and dot blotting for detection of gene expression at the transcriptionlevel; ELISA, radioimmunoassays, immunoblotting, immunoprecipitation,and flow cytometry for detection of gene expression at the translationlevel.

Samples that may be used for measuring such an index include tissues andfluids obtained from an individual, such as blood, obtained in a biopsy,and a fecal sample.

<Method for Predicting Response of an Individual to the BacteriaComposition by Monitoring the Individual's Response to Treatment withthe Composition>

Also described is a method in which an amount (e.g. count) or thepercentage of at least one bacterial species selected from the groupconsisting of: Clostridium saccharogumia, Clostridium ramosum JCM1298,Clostridium ramosum, Flavonifractor plautii, Pseudoflavonifractorcapillosus ATCC 29799, Clostridium hathewayi, Clostridiumsaccharolyticum WM1, Bacteroides sp. MANG, Clostridium saccharolyticum,Clostridium scindens, Lachnospiraceae bacterium 5_1_57FAA,Lachnospiraceae bacterium 6_1_63FAA, Clostridium sp. 14616, Clostridiumbolteae ATCC BAA-613, cf. Clostridium sp. MLG055, Erysipelotrichaceaebacterium 2_2_44A, Clostridium indolis, Anaerostipes caccae, Clostridiumbolteae, Lachnospiraceae bacterium DJF_VP30, Lachnospiraceae bacterium3_1_57FAA_CT1, Anaerotruncus colihominis, Anaerotruncus colihominis DSM17241, Ruminococcus sp. ID8, Lachnospiraceae bacterium 2_1_46FAA,Clostridium lavalense, Clostridium asparagiforme DSM 15981, Clostridiumsymbiosum, Clostridium symbiosum WAL-14163, Eubacterium contortum,Clostridium sp. D5, Oscillospiraceae bacterium NML 061048, Oscillibactervalericigenes, Lachnospiraceae bacterium A4, Clostridium sp. 316002/08,and Clostridiales bacterium 1_7_47FAA, Blautia cocoides, Anaerostipescaccae DSM 14662 in a patient's sample (e.g. a colonic biopsy or a fecalsample) is determined. When the percentage or the count of the bacteriaselected from the list above is lower in an individual than a base linevalue obtained by performing a similar determination on a healthyindividual (e.g., an individual who does not have/has not beenidentified as having a disease or condition for which the bacterialcomposition is a potential treatment such as an auto-immune disease, anallergic condition, cancer, organ rejection), it is determined that theindividual is likely to be responsive to the bacterial composition. Thisdetermination can be used, for example, by a clinician to determinewhether an individual or a patient is likely to benefit from treatmentwith the bacterial composition, or to select an individual or a patientfor inclusion in a clinical trial. The clinician can then administer thebacterial composition to the individual or patient based on thedetermination that the individual or patient is likely to benefit fromtreatment. This determination can also be used as a method to monitor anindividual's response to treatment with the bacterial compositionsdescribed, wherein a higher value of the determination after treatmentwith the bacterial composition (compared to a determination beforetreatment) indicates that the individual has responded favorably totreatment (e.g. is a positive indicator of successful colonization andenhanced immunosuppression in the individual). Optionally, the prognosisand monitoring methods described here may further comprise the step ofmeasuring in the individual's samples the percentages or absolute countsof other commensal species belonging to Clostridium Clusters IV and XIVathat are not present in the bacterial composition, wherein lower thanbaseline values before treatment indicate a higher likelihood of apositive response to treatment, and wherein an increased value aftertreatment indicates that the individual has responded favorably totreatment. In the prognosis and monitoring methods described here, avariety of known methods can be used for determining the composition ofthe microbiota. For example, 16S rRNA sequencing can be used

<Vaccine Adjuvant Composition and Method for Treating or PreventingInfectious Disease or Autoimmune Disease by Using the VaccineComposition>

As described above, and as shown in the Examples, the induction of Tregcells in the colon by bacteria belonging to the Clostridia class has animportant role in local and systemic immune responses. The bacterialcomposition can also be used as an adjuvant to improve the efficacy of avaccine formulation. In one embodiment, the bacterial composition can beused as an adjuvant to a vaccine for the prophylaxis or treatment of anautoimmune disease or an allergic disease (for example, as an adjuvantfor a vaccination protocol where the amount of an allergen is graduallyincreased).

Example of autoimmune diseases and allergic diseases include thosedescribed as the “specific examples of target diseases” in <CompositionHaving Effect of Inducing Proliferation or Accumulation of Regulatory Tcells>.

Other Embodiment

The bacterial composition can also be administered to an individual whois also receiving antibiotic treatment. The present inventors havedemonstrated that antibiotics that act against Gram+ bacteria, such asvancomycin or metronidazole, can effectively eliminate or greatly reducebacterial species belonging to the Clostridia class from thegastrointestinal tract of mammals and subsequently decrease the levelsof regulatory T cells (Example 5). Without wishing to be bound bytheory, the key role of bacteria belonging to the Clostridia class inpreserving immune tolerance strongly indicates that their absence orreduced levels can play a key role in autoimmune diseases characterizedby failures of immune tolerance. Accordingly, individuals undergoingcourses of antibiotics against Gram+ bacteria (for example, individualsbeing treated for infections with pathogens such as C. difficile andGiardia), who are at a high risk of experiencing a loss of the bacteriabelonging to the Clostridia class and thus experience immune tolerancedeficits, can be preventively “repopulated” through use of the bacterialcomposition. The bacterial composition can be administered before,simultaneously with, or after the antibiotic treatment, but preferablyit is administered simultaneously or after the antibiotic treatment. Thebacterial composition is preferably administered in spore form, toimprove its resistance to residual antibiotics. Antibiotics againstGram-positive bacteria include, but are not limited to, vancomycin,metronidazole, linezolid, ramoplanin, fidaxomicin, cephalosporinantibiotics (cephalexin, cefuroxime, cefadroxil, cefazolin, cephalothin,cefaclor, cefamandole, cefoxitin, cefprozil, and ceftobiprole);fluoroquinolone antibiotics (cipro, Levaquin, floxin, tequin, avelox,and norflox); tetracycline antibiotics (tetracycline, minocycline,oxytetracycline, and doxycycline); penicillin antibiotics (amoxicillin,ampicillin, penicillin V, dicloxacillin, carbenicillin, vancomycin, andmethicillin); and carbapenem antibiotics (ertapenem, doripenem,imipenem/cilastatin, and meropenem).

<Methods to Select Treg-Inducing Organisms>

Also described is a method of obtaining bacteria capable of inducingTregs, comprising (1) isolating the bacterial spore-forming fractionfrom a fecal or biopsy sample obtained from a mammal, preferably a human(e.g. by chloroform treatment or by heat treatment), (2) optionally,orally administering the spore-forming fraction to a non-human mammal,preferably a germ-free non-human mammal; (3) optionally, obtaining afecal sample from the non-human mammal, diluting the fecal sample (forexample diluting it by volume by a factor of 10, 100, 1,000, or 10,000),thereby producing a diluted fecal sample, and orally administering thediluted sample to a second germ-free non-human mammal, wherein optionalstep (3) can be repeated more than one time, (4) plating serialdilutions, under aerobic condition or strictly anaerobic conditions, ofeither the spore-forming fraction obtained in (1) or a sample ofintestinal contents of the non-human mammal of (3), and (5) picking asingle colony from the culture plate. The colony can be further assessedfor the ability of bacteria to induce proliferation of regulatory Tcells and/or accumulation of regulatory T cells using known methods,such as those described in the examples.

Following are examples, which describe specific aspects. They are notintended to be limiting in any way.

Note that mice used in Examples were prepared or produced as follows. Inthe following description, mice may be referred to as “SPF” or “GF”.These “SPF” and “GF” indicate that the mice were maintained in theabsence of specific pathogenic bacteria (specific pathogen-free, SPF),and that the mice were maintained under Germ-Free (GF) conditions,respectively.

<Mice>

C57BL/6, Balb/c, and IQI mice maintained under SPF or GF conditions werepurchased from Sankyo Labo Service Corporation, Inc. (Japan), JAPAN SLC,INC. (Japan), CLEA Japan, Inc. (Japan), or The Jackson Laboratory (USA).GF mice and gnotobiotic mice were bred and maintained within thegnotobiotic facility of The University of Tokyo, Yakult CentralInstitute for Microbiological Research, or Sankyo Labo ServiceCorporation, Inc. Myd88^(−/−), Rip2^(−/−), and Card9^(−/−) mice wereproduced as described in NPL 1 to 3, and backcrossed for 8 generationsor more, so that a C57BL/6 genetic background was achieved. Foxp3^(eGFP)mice were purchased from the Jackson Laboratory.

<I110^(venus) Mice>

To form a bicistronic locus encoding both I110 and Venus under controlof an I110 promoter, a targeting construct was first created.Specifically, a cassette (IRES-Venus-SV40 polyA signal cassette, referto Non-Patent Document 4) which was made of an internal ribosome entrysite (IRES), a yellow fluorescent protein (Venus), and a SV40 polyAsignal (SV40 polyA) and which was arranged next to a neomycin-resistantgene (neo), was inserted between a stop codon and a polyA signal (Exon5) of a I110 gene. Next, the obtained targeting construct was used tocause homologous recombination with the I110 gene region in the genomeof mice. Thus, I110^(venus) mice having an I110^(venus) alleles wereproduced (refer to FIG. 1). Note that in FIG. 1 “tk” represents a genecoding thymidine kinase, “neo” represents the neomycin-resistant gene,and “BamH1” represents a cleavage site by the restriction enzyme BamH1.

Genomic DNAs were extracted from the I110^(venus) mice, treated withBamH1, and Southern blotted by use of a probe shown in FIG. 1. FIG. 2shows the obtained results. Wild-type and I110^(venus) alleles weredetected as bands having sizes of 19 kb and 5.5 kb, respectively. Hence,as is apparent from the results obtained, the homologous recombinationoccurred in the genome of the I110^(venus) mice.

Further, CD4⁺ Venus⁻ cells or CD4⁺ Venus⁺ cells in the colonic laminapropria of the I110^(venus) mice were sorted by use of a FACSAria. Then,real-time RT-PCR was carried out on an ABI 7300 system by a method to bedescribed later, to determine the amount of IL-10 mRNA expressed. It wasfound that, since the development of the IL-10 mRNA was detected only inthe CD4⁺ Venus⁺ cells, the expression of IL-10 mRNA in the I110^(venus)mice was correctly reflected in the expression of Venus. Note that thegerm-free states of such I110^(venus) mice were established in CentralInstitute for Experimental Animals (Kawasaki, Japan). The I110^(venus)mice in the germ-free states were maintained in vinyl isolators inSankyo Labo Service Corporation, Inc. (Tokyo, Japan), and used in thefollowing Examples.

Experiments and analyses in Examples were carried out as follows.

<Method for Colonization of Mice with Murine Bacteria and AnalysisThereof>

According to the description in NPL 5 and 6, mice in which SFB orClostridium were colonized were produced. Cecal contents or feces of theobtained gnotobiotic mice were dissolved in sterile water or ananaerobic dilution solution. The dissolved cecal contents or feces asthey were or after a chloroform treatment were orally administered to GFmice. Three strains of the Lactobacillus and 16 strains of theBacteroides were cultured separately from each other in a BL or EG agarmedium in an anaerobic manner. The cultured bacteria were harvested,suspended in an anaerobic TS broth, and orally administrated forcibly toGF mice. The state of the colonization of the bacteria in the mice wasassessed by microscopic observation conducted on a smear preparation offecal pellets.

<Isolation of Intestinal Lamina Propria Lymphocytes and Flow Cytometry>

The small intestine and colon were collected and opened longitudinally.The cecum was also isolated and cecal content was directly frozen at−80° C. or suspended in 2 ml PBS, then added 40% glycerol (finalconcentration 20%), snap-frozen in liquid nitrogen and stored at −80° C.until use. The colon and small intestine were washed in PBS to removeall luminal contents and shaken in Hanks' balanced salt solution (HBSS)containing 5 mM EDTA for 20 min at 37° C. After removing epithelialcells, muscle layers and fat tissue using tweezers, the lamina proprialayers were cut into small pieces and incubated with RPMI1640 containing4% fetal bovine serum, 1 mg/ml collagenase D, 0.5 mg/ml dispase and 40μg/ml DNase I (all Roche Diagnostics) for 1 h at 37° C. in a shakingwater bath. The digested tissues were washed with HBSS containing 5 mMEDTA, resuspended in 5 ml of 40% Percoll (GE Healthcare) and overlaid on2.5 ml of 80% Percoll in a 15-ml Falcon tube. Percoll gradientseparation was performed by centrifugation at 800 g for 20 min at 25° C.The lamina propria lymphocytes were collected from the interface ofPercoll gradient and suspended in ice-cold PBS. For analysis ofregulatory T cells, isolated lymphocytes were labeled with the LIVE/DEADfixable violet dead cell stain kit (Invitrogen) to exclude dead cells inthe analysis. The cells were washed with staining buffer containing PBS,2% FBS, 2 mM EDTA and 0.09% NaN3 and stained surface CD4 withPECγ7-labeled anti-CD4 Ab (RM4-5, BD Biosciences). Intracellularstaining of Foxp3 and Helios was performed using the Alexa700-labeledanti-Foxp3 Ab (FJK-16s, eBioscience), Alexa647-labeled anti-Helios(22F6, eBioscience) and Foxp3 Staining Buffer Set (eBioscience). Foranalysis of Th1 and Th17 cells, isolated lymphocytes were stimulated for4 hours with 50 ng/ml phorbol 12-myristate 13-acetate (PMA, Sigma) and 1μg/ml ionomycin (Sigma) in the presence of GolgiStop (BD Biosciences).After incubation for 4 hours, cells were washed in PBS, labeled with theLIVE/DEAD fixable violet dead cell stain kit and stained surface CD4with PECγ7-labeled anti-CD4 Ab. Cells were washed, fixed inCytofix/Cytoperm, permeabilized with Perm/Wash buffer (BD Biosciences),and stained with the APC-labeled anti-IL-17 Ab (eBio17B7, eBioscience)and FITC-labeled anti-IFN-γ Ab (XMG1.2, BD Biosciences). The Ab stainedcells were analyzed with a LSR Fortessa (BD Biosciences), and data wereanalyzed using Flow Jo software (Treestar).

<Real-Time RT-PCR>

From an RNA prepared by using RNeasy Mini Kit (Qiagen), a cDNA wassynthesized by use of a MMV reverse transcriptase (Promega KK). The cDNAobtained was analyzed by real-time RT-PCR using Power SYBR Green PCRMaster Mix (Applied Biosystems) and ABI 7300 real time PCR system(Applied Biosystems), or real-time RT-PCR using SYBR Premix Ex Taq(TAKARA) and Light Cycler 480. For each sample, a value obtained wasnormalized for the amount of GAPDH. A primer set was designed by usingPrimer Express Version 3.0 (Applied Biosystems), and those exhibiting a90% or higher sequence identity at an initial evaluation were selected.The primer set used was as follows:

Foxp3 (SEQ ID NO: 1) 5′-GGCAATAGTTCCTTCCCAGAGTT-3′ (SEQ ID NO: 2)5′-GGGTCGCATATTGTGGTACTTG-3′ CTLA4 (SEQ ID NO: 3)5′-CCTTTTGTAGCCCTGCTCACTCT-3′ (SEQ ID NO: 4) 5′-GGGTCACCTGTATGGCTTCAG-3′GITR (SEQ ID NO: 5) 5′-TCAGTGCAAGATCTGCAAGCA-3′ (SEQ ID NO: 6)5′-ACACCGGAAGCCAAACACA-3′ IL-10 (SEQ ID NO: 7)5′-GATTTTAATAAGCTCCAAGACCAAGGT-3′ (SEQ ID NO: 8)5′-CTTCTATGCAGTTGATGAAGATGTCAA-3′ GAPDH (SEQ ID NO: 9)5′-CCTCGTCCCGTAGACAAAATG-3′ (SEQ ID NO: 10) 5′-TCTCCACTTTGCCACTGCAA-3′Mmp2 (SEQ ID NO: 11) 5′-GGACATTGTCTTTGATGGCA-3′ (SEQ ID NO: 12)5′-CTTGTCACGTGGTGTCACTG-3′ Mmp9 (SEQ ID NO: 13)5′-TCTCTGGACGTCAAATGTGG-3′ (SEQ ID NO: 14) 5′-GCTGAACAGCAGAGCCTTC-3′Mmp13 (SEQ ID NO: 15) 5′-AGGTCTGGATCACTCCAAGG-3′ (SEQ ID NO: 16)5′-TCGCCTGGACCATAAAGAA-3′ Ido1 (SEQ ID NO: 17)5′-AGAGGATGCGTGACTTTGTG-3′ (SEQ ID NO: 18) 5′-ATACAGCAGACCTTCTGGCA-3′.

<Preparation and Culturing of Large Intestinal Epithelial Cells (IECs)>

First, the colon was collected, cut open longitudinally, and rinsed withPBS. Subsequently, the colon was treated with 1 mM dithiothreitol (DTT)at 37° C. for 30 minutes on a shaker, and then vortexed for one minuteto disrupt the epithelial integrity. The released intestinal epithelialcells (IECs) were collected, and suspended in 5 ml of 20% percoll. Thesuspension was overlayered on 2.5 ml of 80% percoll in a 15-ml Falcontube. Then, the tube was centrifuged at 25° C. and 780 g for 20 minutesto conduct cell separation by percoll density gradient centrifugation.Cells at the interface were collected, and used as colonic IECs (purity:90% or higher, viability: 95%). The IECs obtained collected weresuspended in RPMI containing 10% FBS, and 1×10⁵ cells of the IECs werecultured in a 24-well plate for 24 hours. Thereafter, the culturesupernatant was collected, and measured for active TGF-β1 level by ELISA(Promega).

Meanwhile, for culturing T cells in vitro, 1.5×10⁵ MACS-purified splenicCD4⁺ T cells were cultured in each well of a round-bottomed 96-wellplate, together with a 50% conditioned medium in which IECs isolatedfrom GF mice or Clostridium-colonized mice were cultured, and with 25ng/ml of hIL-2 (Peprotech), in the presence or absence of 25 μg/ml of ananti-TGF-β antibody (R&D). Note that 10 μg/ml of an anti-CD3 antibodyand an anti-CD28 antibody (BD Bioscience) were bound to theround-bottomed plate. After a 5-day culture, the CD4⁺ T cells werecollected, and subjected to a real-time PCR.

<Colitis Experimental Model>

A fecal suspension from Clostridium-colonized mice was orallyadministered to C57BL/6 mice (2-week old), which were grown in aconventional environment for six weeks.

For preparing a DSS-induced colitis model, 2% (wt/vol) DSS (reagentgrade, DSS salt, molecular weight=36 to 50 kD, manufactured by MPBiomedicals), together with drinking water, was given to the mice forsix days.

Meanwhile, for preparing an oxazolone-induced colitis model, the micewere presensitized by transdermally applying, onto the mice, 150 μl of a3% oxazolone (4-ethoxymethylene-2-phenyl-2-oxazolin-5-one,Sigma-Aldrich)/100% ethanol solution. Five days after that, 150 μl of a1% oxazolone/50% ethanol solution was intrarectally administered againto the presensitized mice under a light anesthesia. Note that theintrarectal administration was conducted by using a 3.5 F catheter.

Each mouse was analyzed daily for body weight, occult blood, bleedingvisible with the naked eyes (gross blood), and the hardness of stool.Moreover, the body weight loss percentage, intestinal bleeding (nobleeding, occult blood (hemoccult+), or bleeding visible with the nakedeyes), and the hardness of stool (normal stool, loose stool, ordiarrhea) were evaluated numerically, and the disease activity index(DAI) was calculated in accordance with the description in “S. Wirtz, C.Neufert, B. Weigmann, M. F. Neurath, Nat Protoc 2, 541 (2007).”

<OVA Specific IgE Reaction>

BALB/c SPF mice were inoculated with a fecal suspension fromClostridium-colonized mice (2-week old), and grown in a conventionalenvironment. Then, 1 μg of OVA (grade V, Sigma) and 2 mg of alum (ThermoScientific), 0.2 ml in total, were intraperitoneally injected to themice (at their ages of 4 weeks and 6 weeks). Sera were collected everyweek from the mice at the root of their tail, and OVA-specific IgE wasmeasured by ELISA (Chondrex). Then, at their ages of 8 weeks, spleniccells were collected, inoculated in a 96-well plate at 1×10⁶ cells perwell, and stimulated with OVA (100 μg/ml) for three days. Thereafter,the culture supernatant was collected, and measured for IL-4 and IL-10levels by ELISA (R&D).

<Statistical Analysis>

The difference between control and experimental groups was evaluated bythe Student's t-test.

<Chloroform Treatment and Oral Inoculation with Fecal Samples into GFMice>

Human stool (2 g) from a healthy volunteer (Japanese, male, 29 y old)was suspended with 20 ml phosphate-buffered saline (PBS) and passedthrough a 70 μm cell strainer to eliminate clumps and debris. Then fecalsuspension was mixed with or without chloroform (final concentration3%), and incubated in a shaking water bath for 60 min. The fecalsuspensions without chloroform treatment were orally inoculated intogerm-free (GF) mice (250 μl/mouse). After evaporation of chloroform bybubbling with N2 gas for 30 min, the aliquots containingchloroform-resistant (spore-forming) fraction of human intestinalbacteria were inoculated into IQI GF mice. Each group of ex-GF mice wasseparately kept in a vinyl isolator for 3 or 4 weeks.

<Co-Housing Experiment>

To evaluate whether Treg-inducing human bacteria can be transmittedhorizontally, IQI GF mice were co-housed for 4 weeks with ex-GF micecolonized with chloroform-treated human feces (Example 21 mice) in avinyl isolator (6 mice, designated as mouse #D1 to #D6

<Inoculation with Diluted Cecal Contents into GF Mice>

The frozen cecal content from ex-GF mice inoculated withchloroform-treated human feces (#C 4) was suspended in 10 times volume(w/v) of PBS, passed through a 70 μm cell strainer and treated 3%chloroform. Then the suspension was diluted 2000 (for 4 mice, designatedas mouse #E1 to #E4) or 20000 (for 8 mice, designated as mouse #F1 to#F8)-fold with PBS and orally inoculated into GF IQI mice (2.5×10⁵ or2.5×10⁴ cells/2500/mouse). After 4 weeks, lymphocytes were collectedfrom colon and small intestine and analyzed for Foxp3+ Treg cellproportion and their Helios expression. Cecal contents were frozen andstored at −80° C. until use.

<Re-Colonization Experiments>

The frozen cecal content from ex-GF mice inoculated with 20000-folddilution (#F3, 7 and 8) was suspended in 10 times volume (w/v) of PBS,passed through a 70 μm cell strainer and treated 3% chloroform. Thesuspensions were orally inoculated into GF IQI mice (5, 4 or 4 mice;designated as mouse #G1 to #G5, #H1 to #H4 or #I1 to #I4, respectively).After 4 weeks, colon and small intestine were collected and analyzed forFoxp3+ Treg cell proportion and their Helios expression. Cecal contentswere suspended in 20% glycerol solution, snap-frozen in liquid nitrogenand stored at −80° C.

<Cultured Bacteria-Colonization Experiments>

The glycerol stock of cecal content from #G2 mouse was diluted with PBSand seeded onto BL agar plate. After 48 hours, all bacterial colonieswere collected by scraping the plates with a plate scraper andinoculated into GF IQI mice (4 mice, designated as mouse #K1 to #K 4).Six bacterial strains were isolated from the freeze stock of cecalcontent from #F8 mouse using BL agar plate. These isolated strains wereinoculated into GF IQI mice (4 mice, designated as mouse #J1 to #J4).(Details of the culture method are described below.)

<16S rRNA Gene Quantitative PCR Analysis

Using a QIAamp DNA Stool mini kit (QIAGEN), bacterial genomic DNA wasisolated from the human stool from a healthy volunteer as describedabove (human stool), cecal contents from GF mice gavaged withchloroform-treated human stool (cecal content of B-4 mouse) or fecesfrom SPF ICR mouse (feces of SPF mouse). The isolated DNA was used astemplate for quantitative PCR. The amplification program consisted ofone cycle at 95° C. for 1 min, followed by 50 cycles at 95° C. for 10 sand 60° C. for 30 s. Quantitative PCR analysis was carried out using aLightCycler 480 (Roche). Relative quantity was calculated by the ΔCtmethod and normalized to the amount of total bacteria. The followingprimer sets were used: total bacteria, 5′-GGTGAATACGTTCCCGG-3′(SEQ IDNO.: 45) and 5′-TACGGCTACCTTGTTACGACTT-3′(SEQ ID NO.: 46); Clostridiumcluster XIVa (Clostridium coccoides subgroup),5′-AAATGACGGTACCTGACTAA-3′ (SEQ ID NO.: 47) and5′-CTTTGAGTTTCATTCTTGCGAA-3′(SEQ ID NO.: 48); Clostridium cluster IV(Clostridium leptum) 5′-CCTTCCGTGCCGSAGTTA-3′(SEQ ID NO.: 49) and5′-GAATTA AACCACATACTCCACTGCTT-3′(SEQ ID NO.: 50); Bacteroides,5′-GAGAGGAAGGTCCCCCAC-3′(SEQ ID NO.: 51) and5′-CGCTACTTGGCTGGTTCAG-3′(SEQ ID NO.: 52); Bifidobacterium,5′-CGGGTGAGTAATGCGTGACC-3′ (SEQ ID NO.: 53) and5′-TGATAGGACGCGACCCCA-3′(SEQ ID NO.: 54). Note that mice gavaged withchloroform-treated human stool exhibited high levels of spore-formingbacteria, such as Clostridium clusters XIVa and IV, and a severedecrease of non-spore-forming bacteria, such as Bacteroides andBifidobacterium, compared with the human stool before chloroformtreatment.

<Isolation of DNA from Cecal Contents for 16S rRNA Gene MetasequenceAnalysis>

The cecal contents of A1-1, A2-4, B-4, E-3, E-7, E-8, F-2, G-3, H-3, I-3and J-3 were collected by centrifugation at 5000×g for 10 min at 4° C.,suspended in 10 ml of Tris-EDTA containing 10 mM Tris-HCl and 1 mM EDTA(pH 8), and then used for DNA isolation. Lysozyme (SIGMA, 15 mg/ml) wasadded to the cell suspension. After incubation at 37° C. for 1 h withgentle mixing, a purified achromopeptidase (Wako) was added (final 2000unit/ml) and incubated at 37° C. for 30 min. Then, sodium dodecylsulfate (final 1%) was added to the cell suspension and mixed well.Subsequently, proteinase K (Merck) was added (final 1 mg/ml) to thesuspension and the mixture was incubated at 55° C. for 1 h.

High-molecular-weight DNA was isolated and purified by phenol/chloroformextraction, ethanol, and finally polyethyleneglycol precipitation.

<16S rRNA Gene Metasequence>

An aliquot of the DNA was used for PCR amplification and sequencing ofbacterial 16S rRNA genes. ˜330 bp amplicons, spanning variable region1-2 (V1-2) of the gene were generated by using (i) modified primer 8F(5′-CCATCTCATCCCTGCGTGTCTCCGACTCAG+Barcode+agrgtttgatymtggctcag-3′ (SEQID NO.: 55)) which consists of 454 adaptor sequence (underlined), asample specific, error correcting barcode (10 bases, bold) and theuniversal bacterial primer 8F and (ii) modified primer 338R(5′-CCTATCCCCTGTGTGCCTTGGCAGTCTCAG+tgctgcctcccgtaggagt-3′(SEQ ID NO.:56)) which contains 454 adaptor sequence (underlined) and the bacterialprimer 338R. Polymerase chain reactions were performed for each fecalDNA sample: each 50-μL reaction contained 40 ng of DNA, 5 μl of 10× ExTaq buffer (TAKARA), 5 μl of 2.5 mM dNTP mixture, 0.2 μl Ex Taq and 0.2μM of each primer. PCR conditions consisted of an initial denaturationstep performed at 96° C. for 2 min, followed by 20 cycles ofdenaturation (96° C., 30 s), annealing (55° C., 45 s) and amplification(72° C., 1 min) and final amplification step performed at 72° C. for 10min. Amplicons generated from each sample were subsequently purifiedusing AMPur XP (Beckman Coulter). The amount of DNA was quantified usingQuant-iT Picogreen dsDNA Assay Kit (Invitrogen) and TBS-380miniFluorometer (Turner Biosystems). The amplified DNA were used as templatefor 454 GS Junior (Roche) pyrosequencing. The sequences were performedusing GS Junior Titanium emPCR Kit-Lib-L, GS Junior Titanium SequencingKit and GS Junior Titanium PicoTiterPlate Kit (all Roche) according tothe manufacturer's manuals (GS Junior Titanium Series, emPCRAmplification Method Manual—Lib-L and Sequencing Method Manual).Resulting sequences (3400 reads were produced for each sample) wereclassified into OTU on the basis of sequence similarity (>97% identity).Representative sequences from each OTU were compared with sequences innucleic acid databases (Ribosomal Database Project) using BLAST todetermine the closest relatives. Then, OTUs were classified into specieson the basis of the closest relatives. All data of close relatives andthe number of reads are shown in Table. 1.

<Isolation of Bacterial Strains>

Bacterial strains were isolated from the cecal contents of #F8, #G2, #I1and #K3 by plating serial dilutions of the cecal samples under aerobiccondition or strictly anaerobic conditions (80% N2 10% H2 10% CO2) ontoBL agar (Eiken Chemical) or EG agar plates containing medium with thefollowing components (quantities expressed per liter): Meat extract 500ml; Proteose peptone No. 3 (10.0 g, Difco); Yeast Extract (5.0 g,Difco); Na2HPO4 (4.0 g); D(+)-Glucose (1.5 g); Soluble Starch (0.5 g);L-cystine (0.2 g), L-cysteine-HCl—H2O (0.5 g); Tween80 (0.5 g); BactoAgar (16.0 g, Difco); defibrinated horse blood (50 ml). After culture at37° C. for 2 or 4 days, each single colony was picked up and culturedfor additional 2 or 4 days at 37° C. by ABCM broth or EG agar plate. Theisolated strains were collected into EG stock medium (10% DMSO) andstored at −80° C. For suspension of isolated strains to re-inoculatemice, TS medium (27.5 g of trypticase soy broth w/o dextrose, 0.84 g ofNa2CO3, 0.5 g of L-cysteine-HCl—H2O, 1000 ml of distilled water, pHadjusted to 7.2+/−0.2 with NaOH, then autoclaved for 15 minutes at 115degrees Celsius). To identify the isolated strains, 16SrRNA coding genesequences were performed. The 16S rRNA genes were amplified bycolony-PCR using KOD FX (TOYOBO), 16S rRNA gene-specific primer pairs:8F (5′-AGAGTTTGATCMTGGCTCAG-3′(SEQ ID NO.: 57)) and 519R(5′-ATTACCGCGGCKGCTG-3′(SEQ ID NO.: 58)) for C. indolis, C. bolteae,Bacteroides sp. MANG, L. bacterium DJF_VP30, A. colihominis,Ruminococcus sp. ID8, C. lavalense, C. symbiosum and E. contortum or1513R (5′-ACGGCTACCTTGTTACGACTT-3′(SEQ ID NO.: 59)) for C.saccharogumia, C. ramosum, F. plautii, C. hathewayi, C. scindens,Clostridium sp. 2335, Clostridium sp. 14616 and cf Clostridium sp.MLG055 and GeneAmp PCR System9700 (Applied Biosystems). Theamplification program consisted of one cycle at 98° C. for 2 min,followed by 40 cycles at 98° C. for 10 s, 57° C. for 30 s and 68° C. for40 s. Each amplified DNA was purified from the reaction mixture usingIllustra GFX PCR DNA and Gel Band Purification Kit (GE Healthcare).Sequence analysis was performed using BigDye Terminator V3.1 CycleSequencing Kit (Applied Biosystems) and Applied Biosystems 3730xl DNAanalyzer (Applied Biosystems). The resulting sequences were comparedwith sequences in nucleic acid databases using BLAST to determine theclosest relatives. The closest relatives and % identity of all isolatedstrains, information for genus-species of the closest relatives,Clostridium cluster, ID of mouse from which was derived, maximumsimilarity and culture medium of isolated strains were summarized inTable 2.

Example 1

First, it was investigated whether or not accumulation of regulatory Tcells (Treg cells) in the colonic lamina propria was dependent oncommensal bacteria. Specifically, lymphocytes were isolated fromperipheral lymph nodes (pLN) of Balb/c mice bred in the absence ofspecific pathogenic bacteria (SPF) or from lamina propria of the colonor the small intestine (SI) of the mice. The CD4 and Foxp3 were stainedby antibodies. Then, the ratio of Foxp3⁺ cells in CD4⁺ lymphocytes wasanalyzed by flow cytometry. The results showed that Foxp3⁺ Treg cellswere present at a high frequency in the lamina propria of thegastrointestinal tracts, especially in the colonic lamina propria, ofthe mice kept under the environment free from specific pathogenicmicroorganisms (SPF). In addition, it was also found that the number ofthe Foxp3⁺ Treg cells in the colonic lamina propria gradually increasedup to three months after their birth, whereas the number of the Foxp3⁺Treg cells in the peripheral lymph nodes was basically constant from thetime of two weeks after their birth.

Example 2

Next, it was investigated whether or not the temporal accumulation ofthe Treg cells in the colon as found in Example 1 had a relationshipwith the colonization of intestinal commensal microbiota. Specifically,the expression of CD4 and the expression of Foxp3 in lymphocytesisolated from the small intestine, the colon, and the peripheral lymphnodes of mice bred under a germ-free (GF) or SPF environment (8 weeksold: Balb/c mice, IQI mice, and C57BL/6 mice) were analyzed. Similarresults were obtained in three or more independent experiments.

In addition, lamina propria lymphocytes were collected from SPF mice andGF mice (Balb/c mice or C57BL/6 mice). CD4 and Foxp3 were stained withantibodies. Then, the lamina propria lymphocytes were analyzed by FACS.

Further, lymphocytes were isolated from the lamina propria of the colon,the lamina propria of the small intestine (SI), Peyer's patches (PPs),and mesenteric lymph nodes (MLNs) of mice (SPF C57BL/6 mice) to whichantibiotics were orally administered with water for eight weeks. CD4 andFoxp3 were stained with antibodies. Then, the lymphocytes were analyzedby FACS. Similar results (the ratio of the Foxp3⁺ cells in the CD4⁺cells of an individual mouse) were obtained in two or more independentexperiments. Note that the following antibiotics were used incombination in accordance with the description in the followingdocument:

ampicillin (A; 500 mg/L, Sigma)

vancomycin (V; 500 mg/L, NACALAI TESQUE, INC.)

metronidazole (M; 1 g/L, NACALAI TESQUE, INC.)

neomycin (N; 1 g/L, NACALAI TESQUE, INC.)

-   Rakoff-Nahoum, J. Paglino, F. Eslami-Varzaneh, S. Edberg, R.    Medzhitov, Cell 118, 229 (Jul. 23, 2004)-   Fagarasan et al., Science 298, 1424 (Nov. 15, 2002)

As is apparent from the results the frequencies and the absolute numbersof Foxp3⁺ CD4⁺ cells in the small intestine and the peripheral lymphnodes of the GF mice were equal to or greater than those of the SPFmice. In addition, the numbers of the Treg cells in the small intestinallamina propria, Peyer's patches, and mesenteric lymph nodes of the SPFmice to which the antibiotics were orally administered for eight weekswere equal to or greater than those of the SPF mice that had notreceived antibiotics. Meanwhile, the number of the Foxp3⁺ CD4⁺ cells inthe colonic lamina propria of the GF mice was decreased significantly incomparison with that of the SPF mice. This decrease was commonlyobserved among mice of different genetic backgrounds (Balb/c, IQI, andC57BL/6), as well as among mice bred in different animal facilities. Inaddition, it was also shown that the number of Treg cells in the coloniclamina propria of the SPF C57BL/6 mice to which the antibiotics wereadministered was decreased significantly.

Example 3

Next, it was directly checked whether or not the decrease in the numberof the Treg cells in the colonic lamina propria of the GF mice shown inExample 2 was attributed to the absence of microbiota. Specifically, afecal suspension of B6 SPF mice purchased from The Jackson Laboratorywas orally administered to GF-IQI mice (conventionalization). Threeweeks after the administration, lymphocytes were isolated from thecolonic lamina propria, and the expression of Foxp3 in CD4⁺ lymphocyteswas analyzed. The results showed that the number of Treg cells in thesmall intestinal lamina propria did not change. However, the number ofthe Treg cells in the colonic lamina propria increased significantly.Hence, it was shown that host-microbial interaction played an importantrole in the accumulation of Foxp3⁺ Treg cells in the colonic laminapropria, while the accumulation of the Treg cells in the smallintestinal lamina propria had a different mechanism.

Example 4

Next, the relationship between the gut-associated lymphoid tissues ofmice and the number of Foxp3⁺ cells in the colonic lamina propria of themice was investigated in accordance with the method described in M. N.Kweon et al., J Immunol 174, 4365 (Apr. 1, 2005). Specifically, 100 μgof an extracellular domain recombinant protein (a fusion protein(LTβR-Ig) between a lymphotoxin β receptor (LTβR) and a Fc region ofhuman IgG1, refer to Honda et al., J Exp Med 193, 621 (Mar. 5, 2001))was injected intraperitoneally into pregnant C57BL/6 mice 14 days afterconception. The LTβR-Ig was again injected intraperitoneally intofetuses obtained from such mice, so that mice from which isolatedlymphoid follicles (ILFs), Peyer's patches (PPs), and colonic-patches(CPs) were completely removed were produced. Then, the ratios of Foxp3⁺cells in CD4⁺ cells in the colonic lamina propria of the mice treatedwith the LTβR-Ig, and mice treated with rat IgG (control) were analyzedby FACS. The results show that the ratio of the Foxp3⁺ cells in thecolonic lamina propria of the mice deficient in isolated lymphoidfollicles, Peyer's patches, and the colonic-patches (the mice treatedwith the LTβR-Ig) rather increased. Accordingly, it was suggested thatthe decrease in the number of the Treg cells in the colonic laminapropria of the GF mice and the mice treated with the antibiotics wascaused because the transmission of specific signals which promotes theaccumulation of Treg cells in the colonic lamina propria and which iscaused by the intestinal microbes did not occur, rather than simplybecause of a secondary effect of disorganized gut-associated lymphoidtissues.

Example 5

To investigate whether or not a specific intestinal flora induced theaccumulation of colonic Treg cells, vancomycin as an antibiotic againstGram-positive bacteria or polymyxin B as an antibiotic againstGram-negative bacteria was administered to SPF mice (from 4 weeks ofage) for four weeks, and analyzed for the ratio of Foxp3⁺ cells in theCD4⁺ cell group ([%] Foxp3⁺ in CD4).

The results show that the number of Treg cells in the colon of the miceto which vancomycin was administered was markedly decreased incomparison with that of the control. In contrast, no influence wasobserved on the number of Treg cells of the mice to which polymyxin Bwas administered. Those facts suggested that Gram-positive commensalbacteria played a major role in accumulation of Treg cells.

Example 6

A recent report has suggested that spore-forming bacteria play animportant role in intestinal T cells response (see V. Gaboriau-Routhiauet al., Immunity 31, 677 (Oct. 16, 2009)). In this respect, fecalmicroorganisms (spore-forming fraction) resistant to 3% chloroform wereorally administered to GF mice, which were then analyzed for the ratioof Foxp3⁺ cells in the CD4⁺ cell group ([%] Foxp3⁺ in CD4).

Three weeks after the administration of the chloroform-treated feces,the number of Treg cells in the administered mice was markedly increasedto the same level as those of the SPF mice and the GF mice to which theuntreated feces was forcibly administered.

Accordingly, considering the results shown in Example 5 in combination,it was revealed that the specific components of the indigenousmicrobiota were highly likely to belong to the Gram-positive group, andthat the spore-forming fraction played an important role in theinduction of Treg cells.

Example 7

Next, the species of the intestinal microbiota which induced theaccumulation of Treg cells in the colon as suggested in Examples 4 to 6were identified. Specifically, segmented filamentous bacteria (SFB), 16strains of the Bacteroides spp. (Bactero. (6 strains of B. vulgatus, 7of the B. acidifaciens group 1, and 3 of the B. acidifaciens group 2)),3 strains of the Lactobacillus (Lacto. (L. acidophilus, L. fermentum,and L. murinum)), and 46 strains of Clostridium spp. (Clost., refer to“Itoh, K., and Mitsuoka, T. Characterization of clostridia isolated fromfaeces of limited flora mice and their effect on caecal size whenassociated with germ-free mice. Lab. Animals 19: 111-118 (1985))”), ormicrobiota collected from mice (SPF) bred under a conventionalenvironment was orally administered to GF-Balb/c mice or GF-IQI mice.The mice were maintained in vinyl isolators for three weeks. Then, CD4cells were isolated from the colon and the small intestine of thesemice. The numbers of Treg cells in the colon and the small intestinewere analyzed by flow cytometry.

The bacteria belonging to the genus Clostridium are classified bysequencing of 16S rRNA gene, as follows. Specifically, the 16S rRNAgenes of the bacteria were amplified by PCR using 16S rRNA gene-specificprimer pairs: 5′-AGAGTTTGATCMTGGCTCAG-3′ (SEQ ID NO: 60) and5′-ATTACCGCGGCKGCTG-3′ (SEQ ID NO: 61) (see T. Aebischer et al.,Vaccination prevents Helicobacter pylori-induced alterations of thegastric flora in mice. FEMS Immunol. Med. Microbiol. 46, 221-229(2006)). The 1.5-kb PCR product was then introduced into pCR-BluntVector. The inserts were sequenced and aligned using the ClustalWsoftware program. The resulting sequences of 16S rRNA genes derived fromstrain 1-41 of 46 strains of Clostridium spp. were shown in SEQ ID NO:21-61. A phylogenetic tree was constructed by the neighbor-joiningmethod with the resulting sequences of the 41 strains of Clostridium andthose of known bacteria obtained from Genbank database using Megasoftware.

The results showed no effect on the number of the Treg cells in thecolon was observed in the GF mice in which the segmented filamentousbacteria (SFB) were colonized. Moreover, mice in which the cocktail ofthree strains of Lactobacillus was colonized gave similar results. Onthe other hand, it was shown that the accumulation of Foxp3⁺ cells inthe colonic lamina propria was strongly induced in the mice in which 46strains of Clostridium spp. were colonized. Importantly, suchaccumulation was promoted irrespective of the genetic backgrounds of themice, and led to the increase in number similar to that in the SPF micealthough intestinal microbiota of only a single genus were colonized. Itwas also shown that the colonization of the Clostridium did not changethe number of Treg cells in the small intestinal lamina propria. Notethat, when the 16 strains of Bactericides spp. were colonized, thenumber of Treg cells in the colon was increased significantly. However,the extent of the increase varied depending on the genetic background ofthe mice in which the bacteria were colonized.

Example 8

Next, CD4 expression, Foxp3 expression, and Helios expression in LPlymphocytes of the thymuses and the colons of SPF mice, GF mice,Lactobacillus-colonized mice, and Clostridium-colonized mice wereanalyzed by flow cytometry.

The results show that most Foxp3⁺ cells found in the SPF mice or theClostridium-colonized mice did not express Helios. Note that Helios is atranscription factor known to be expressed in thymic-derived naturalTreg cells (see A. M. Thornton et al., J Immunol 184, 3433 (Apr. 1,2010)). Accordingly, it was suggested that most of the Treg cells in theSPF mice and the Clostridium-colonized mice were Treg cells induced inperipheral portions (so-called iTreg cells).

Example 9

Next, it was investigated whether or not the colonization of theClostridium or the like had an influence on other T cells. Specifically,SFB, 16 strains of Bacteroides spp. (Bactero.), 46 strains ofClostridium spp. (Clost.), or microbiota collected from mice bred undera conventional environment (SPF) was colonized in GF IQI mice. Threeweeks later, lymphocytes in the colonic lamina propria were isolatedfrom these mice, and stimulated with PMA (50 ng/ml) and ionomycin (1μg/ml) for four hours in the presence of Golgistop (BD Bioscience).After the stimulation was given, intracellular cytokines were stained byusing an anti-IL-17 PE antibody (TC11-18H10) and an anti-IFN-g FITCantibody (BD Bioscience) in accordance with the manual of acytofix/cytoperm kit (BD Bioscience). Then, the ratio of IFN-γ⁺ cells orIL-17⁺ cells in CD4⁺ leucocytes was analyzed by flow cytometry. Theresults show that the colonization of the Clostridium did not have anyinfluence on Th1 cells (CD4⁺ IFN-γ⁺ cells) in the colon, and caused onlya slight increase of Th17 cells (CD4⁺ IL-17⁺ cells). Accordingly, it wassuggested that the genus Clostridium was a genus of bacteria whichspecifically induced Treg cells.

Example 10

It has been reported that 46 strains of Clostridium spp. exert aninfluence on the accumulation of CD8⁺ intestinal tract intraepitheliallymphocytes (IELs) in the colon. Accordingly, it is conceivable thatClostridium regulates the immune system in various aspects, and thatClostridium exhibits a marked ability to induce and maintain Treg cellsespecially in the colon, as described above. In addition, a kind ofcytokines, transforming growth factor-β (TGF-β), is known to play animportant role in regulation of Treg cell generation.

In this respect, it was examined whether or not the colonization ofClostridium provided a colonic environment rich in TGF-β. Specifically,first, the whole colons of GF mice, Clostridium-colonized mice, andLactobacillus-colonized mice were cultured for 24 hours, and the culturesupernatants thereof were measured for the concentration of active TGF-β(TGF-β1) by ELISA (the number of mice analyzed was four per group).

The results show that the amount of TGF-β produced in the colons of theClostridium-colonized mice was significantly greater than that in colonsof the GF mice and the Lactobacillus-colonized mice.

Next, intestinal epithelial cells (IECs) of GF mice andClostridium-colonized mice were cultured for 24 hours, and the culturesupernatants thereof were measured for the concentration of active TGF-β(TGF-β1) by ELISA (the number of mice analyzed was four per group).

The results show that TGF-β was detected in the culture supernatant ofthe IECs isolated from the Clostridium-colonized mice, whereas no TGF-βwas detected in the culture supernatant of the IECs isolated from the GFmice.

Next, as described above, splenic CD4⁺ T cells were cultured for fivedays together with a 50% conditioned medium in which IECs isolated fromthe GF mice or the Clostridium-colonized mice were cultured, and withthe anti-CD3 antibody, in the presence or absence of an anti-TGF-βantibody. Then, the T cells were collected, and analyzed for expressionof Foxp3 by real-time RT-PCR.

The results show that when the culture supernatant of the IECs derivedfrom the Clostridium-colonized mice was added to the splenic CD4⁺ Tcells, differentiation into Foxp3-expressing cells was accelerated.Meanwhile, differentiation into Treg cells was inhibited by theanti-TGF-β antibody.

The expression of MMP2, MMP9, and MMP13, which are thought to contributeto the activation of latent TGF-β was investigated. The expression ofindoleamine 2,3-dioxygenase (IDO), which is thought to be involved inthe induction of Treg cells, was also investigated. Specifically, 46bacterial strains of the genus Clostridium (Clost.), or three bacterialstrains of the genus Lactobacillus (Lacto.) were orally administered toC57BL/6 germ-free mice. Three weeks after administration, IECs werecollected, and analyzed for relative mRNA expression levels of MMP2,MMP9, MMP13, and IDO genes by real-time RT-PCR (the number of miceanalyzed was three per group).

For the relationship between the activation of latent TGF-β and theabove-describe MMP, see D'Angelo et al., J. Biol. Chem. 276,11347-11353, 2001; Heidinger et al., Biol. Chem. 387, 69-78, 2006; Yu etal., Genes Dev. i4, 163-176, 2000. For the relationship between IDO andthe induction of Treg cells, see G. Matteoli et al., Gut 59, 595 (May,2010).

The results show in agreement with the production of TGF-β describedabove, that transcription products of the genes encoding MMP2, MMP9, andMMP13 were expressed at higher levels in the IECs derived from theClostridium-colonized mice than in those in the GF mice and in theLactobacillus-colonized mice.

Moreover, IDO was expressed only in the Clostridium-colonized mice.

Accordingly, it was revealed that the Clostridium activated the IECs,and led to the production of TGF-β and other Treg cell-inducingmolecules in the colon.

Example 11

Next, it was investigated whether or not the Treg cell accumulationinduced by the colonization of the Clostridium was dependent on signaltransmission by pathogen-associated molecular pattern recognitionreceptors. Specifically, the numbers of Treg cells in the colonic laminapropria of each SPF mice of Myd88^(−/−) (deficient in Myd88 (signalingadaptor for Toll-like receptor)), Rip2^(−/−) (deficient in Rip2 (NODreceptor adaptor)), and Card9^(−/−) (deficient in Card9 (essentialsignal transmission factor for Dectin-1 signal transmission)) wereexamined. In addition, Clostridium spp. were caused to be colonized inthe Myd88^(−/−) GF mice, and the change in the number of Treg cells wasinvestigated. The results show that the number of Treg cells of eachkind of the SPF mice deficient in the associated factors of thepathogen-associated molecular pattern recognition receptors did notchange relative to that of wild-type mice of the same litter, whichserved as a control. In addition, it was found that when Clostridiumspp. were colonized in GF mice deficient in Myd88, the accumulation ofTreg cells in the colonic lamina propria was induced. Accordingly, ithas been suggested that the mechanism of inducing the accumulation ofTreg cells in the colonic lamina propria relies not on activation ofrecognition pathway for major pathogen-associated molecular patterns asis caused by most bacteria, but on specific commensal bacterial species.

Example 12

Intestinal tract Foxp3⁺ Treg cells are known to exert someimmunosuppressive functions through IL-10 production (refer to NPL 9).Meanwhile, animals having CD4⁺ Foxp3⁺ cells from which IL-10 isspecifically removed are known to develop inflammatory bowel disease(refer to NPL 18). In this respect, first, the expression of IL-10 inlymphocytes of various tissues was examined. Specifically, lymphocyteswere isolated from various tissues of SPF I110^(venus) mice, and theexpression of CD4 and the expression of Venus were analyzed by flowcytometry.

Lymphocytes in the colonic lamina propria were isolated fromI110^(venus) mice, and the expression of T cell receptor β chain (TCRβ)on the surfaces of the cells was detected by FACS.

Lymphocytes in the colonic lamina propria were isolated fromI110^(venus) mice. The lymphocytes were stimulated with PMA (50 ng/ml)and ionomycin (1 μg/ml) for four hours in the presence of Golgistop (BDBioscience). Then, after the stimulation was given, intracellularcytokines were stained by using an anti-IL-17 PE antibody, an anti-IL-4APC antibody (11B11), and an anti-IFN-g FITC antibody (BD Bioscience) inaccordance with the manual of a cytofix/cytoperm kit (BD Bioscience).

In addition, Foxp3⁺ CD4⁺ cells and Foxp3⁻ CD4⁺ cells were isolated fromthe spleen (Sp1) of Foxp3^(eGFP) reporter mice, and Venus⁺ cells wereisolated from the colonic lamina propria and the small intestine (SI)lamina propria of I110^(venus) mice. The obtained cells were analyzed interms of expression of predetermined genes. The gene expression wasanalyzed by real-time RT-PCR using a Power SYBR Green PCR Master Mix(Applied Biosystems) and an ABI 7300 real time PCR system (AppliedBiosystems). Here, the value for each cell was normalized for the amountof GAPDH.

The results show that almost no Venus⁺ cells (IL-10-producing cells)were detected in the cervical lymph nodes (peripheral lymph nodes),thymus, peripheral blood, lung, and liver of mice kept under the SPFconditions. Meanwhile, in the spleen, Peyer's patches, and mesentericlymph nodes thereof, Venus⁺ cells were slightly detected. On the otherhand, many Venus⁺ cells were found in the lymphocytes in the smallintestine lamina propria and colonic lamina propria. In addition, mostof the Venus⁺ cells in the intestines were positive for CD4, and alsopositive for T cell receptor β chain (TCRβ). It was found that theVenus⁺ CD4⁺ T cells expressed Foxp3 and other Treg cell-associatedfactors such as a cytotoxic T-Lymphocyte antigen (CTLA-4) and aglucocorticoid-induced TNFR-associated protein (GITR), although theVenus⁺ CD4⁺ T cells showed none of the phenotypes of Th2(IL-4-producing) and Th17 (IL-17-producing). It was shown that theexpression level of CTLA-4 in the intestinal Venus⁺ cells was higherthan that in the splenic GFP⁺ Treg cells isolated from the Foxp3^(eGFP)reporter mice.

Example 13

Venus⁺ cells can be classified into at least two subsets, namely, Venus⁺Foxp3⁺ double positive (DP) Treg cells and Venus⁺ Foxp3⁻ Treg cells onthe basis of intracellular Foxp3 expression. Cells of the latter subsetcorrespond to type 1 regulatory T cells (Tr1) (refer to NPL 8 and 9). Inthis respect, the Venus⁺ cells (IL-10-producing cells) observed inExample 8 were investigated in terms of the expression of Foxp3.Specifically, the expression of CD4, Foxp3, and Venus in the laminapropria of the colon and the lamina propria of the small intestine ofI110^(venus) mice kept under GF or SPF conditions was analyzed by FACS,and the numbers of Venus⁺ cells in the intestinal tract lamina propriawere compared between SPF and GF I110^(venus) mice.

In addition, the intracellular expression of Venus and Foxp3 in CD4cells in various tissues of SPF I110^(venus) mice was analyzed by flowcytometry.

In order to investigate whether or not the presence of commensalbacteria had any influence on the expression of IL-10 in regulatorycells in the gastrointestinal tracts, germ-free (GF) I110^(venus) micewere prepared. Then, predetermined species of bacteria were caused to becolonized in the obtained GF I110^(venus) mice. Three weeks after thespecies of bacteria were colonized, a CD4⁺ cell group (V⁺F⁻, Venus⁺Foxp3⁻ cells; V⁺F⁺, Venus⁺ Foxp3⁺ cells; and V⁻F⁺, Venus⁻Foxp3⁺ cells)in which Foxp3 and/or Venus were expressed in the colon and the smallintestine was analyzed by flow cytometry.

In order to check whether or not the presence of commensal bacteria hadany influence on the expression of IL-10 in regulatory cells in thegastrointestinal tracts, antibiotics were orally given with water tofive or six I110^(venus) mice per group for 10 weeks. The followingantibiotics were used in combination.

-   -   ampicillin (A; 500 mg/L Sigma)    -   vancomycin (V; 500 mg/L NACALAI TESQUE, INC.)    -   metronidazole (M; 1 g/L NACALAI TESQUE, INC.)    -   neomycin (N; 1 g/L NACALAI TESQUE, INC.)

Then, CD4 and Foxp3 of lymphocytes in the lamina propria of the colon,the lamina propria of the small intestine (SI), mesenteric lymph nodes(MLN), and Peyer's patches (PPs) were stained with antibodies, andanalyzed by FACS. The results were obtained from two or more independentexperiments which gave similar results.

The results show that the small intestinal lamina propria was rich inVenus⁺ Foxp3⁻ cells, namely, Tr1-like cells, and that the Venus⁺ Foxp3⁺DP Treg cells were present at a high frequency in the colon of the SPFmice. In contrast, although sufficient numbers of Foxp3⁺ cells wereobserved also in other tissues, the expression of Venus was not observedin almost all of the cells.

In addition, it was shown that all regulatory T cell fractions of Venus⁺Foxp3⁻, Venus⁺ Foxp3⁺, and Venus⁻ Foxp3⁺ in the colon significantlydecreased under the GF conditions. Moreover, similar decrease in Venus⁺cells was observed also in the SPF I110^(venus) mice treated with theantibiotics.

The colonization of Clostridium spp. strongly induced all regulatory Tcell fractions of Venus⁺ Foxp3⁻, Venus⁺ Foxp3⁺, and Venus⁻ Foxp3⁺ in thecolon, and the degrees of the induction thereof were equal to those inthe SPF mice. In addition, it was found that the colonization of thethree strains of Lactobacillus or the colonization of SFB had anextremely small influence on the number of Venus⁺ and/or Foxp3⁺ cells inthe colon. Moreover, the colonization of 16 strains of Bacteroides spp.also induced Venus⁺ cells, but the influence of the colonization wasspecific to Venus⁺ Foxp3⁻ Tr1-like cells. On the other hand, it wasfound that none of the bacterial species tested exerted any significantinfluence on the number of IL-10-producing cells in the small intestinallamina propria.

Hence, it was shown that the genus Clostridium colonized in the colon ora physiologically active substance derived from the bacteria provided asignal for inducing the accumulation of IL-10⁺ regulatory T cells in thecolonic lamina propria or the expression of IL-10 in T cells. It wasshown that the number of Venus⁺ cells in the small intestine was notsignificantly influenced by the situation where no commensal bacteriawere present or commensal bacteria were decreased, and that IL-10⁺regulatory cells (Tr1-like cells) accumulated in the small intestinallamina propria independently of commensal bacteria.

Example 14

It was investigated whether or not Venus⁺ cells induced by the genusClostridium had an immunosuppressive function similar to that of Venus⁺cells in the colon of SPF mice. Specifically, CD4⁺ CD25⁻ cells (effectorT cells, Teff cells) isolated from the spleen were seeded in aflat-bottomed 96-well plate at 2×10⁴/well, and cultured for three daystogether with 2×10⁴ splenic CD11c⁺ cells (antigen-representing cells)subjected to 30 Gy radiation irradiation treatment, 0.5 μg/ml of ananti-CD3 antibody, and a lot of Treg cells. In addition, for the lastsix hours, the CD4⁺ CD25⁻ cells were cultured, with [³H]-thymidine (1μCi/well) was added thereto. Note that, Treg cells used in Example 14were CD4⁺ GFP⁺ T cells isolated from the spleen of Foxp3^(eGFP) reportermice, or CD4⁺ Venus⁺ T cells in the colonic lamina propria of GFI110^(venus) mice in which Clostridium spp. were colonized or SPFI110^(venus) mice. Then, proliferation of the cells was determined basedon the uptake amount of [³H]-thymidine, and represented by a count perminute (cpm) value.

The results show that Venus⁺ CD4⁺ cells of the mice in which the genusClostridium was colonized suppressed in vitro proliferation of CD25⁻CD4⁺ activated T cells. The suppression activity was slightly inferiorto that of GFP⁺ cells isolated from the Foxp3^(eGFP) reporter mice, butequal to that of Venus⁺ cells isolated from the SPF I110^(venus) mice.Accordingly, it has been shown that the genus Clostridium inducesIL-10-expressing T cells having sufficient immunosuppressive activities,and thereby plays a critical role in maintaining immune homeostasis inthe colon.

Example 15

Next, the influence of the colonization of a large number of Clostridiumon the local immune response and the resultant proliferation of Tregcells were investigated.

<Dextran Sulfate Sodium (DSS)-Induced Colitis Model>

First, the DSS-induced colitis model was prepared as described above,and the influence on the model mice of the inoculation of theClostridium and the proliferation of Treg cells was investigated.Specifically, control mice and Clostridium-inoculated mice were treatedwith 2% DSS, then observed and measured for six days for body weightloss, the hardness of stool, and bleeding, and then were evaluatednumerically. In addition, on day 6, the colons were collected,dissected, and analyzed histologically by HE staining.

The results show that the symptoms of the colitis such as body weightloss and rectal bleeding were significantly suppressed in the micehaving a large number of Clostridium (hereinafter also referred to as“Clostridium-abundant mice”) in comparison with the control mice(C57BL/6 mice grown in a conventional environment for six weeks and notinoculated with the fecal suspension). All the features typical forcolonic inflammation, such as shortening of the colon, edema, andhemorrhage, were observed markedly in the control mice in comparisonwith the Clostridium-abundant mice. Moreover, histological features suchas mucosal erosion, edema, cellular infiltration, and crypt loss wereless severe in the DSS-treated Clostridium-abundant mice than in thecontrol mice.

<Oxazolone-Induced Colitis Model>

Next, the oxazolone-induced colitis model was prepared as describedabove, and the influence on the model mice of the inoculation ofClostridium and the proliferation of Treg cells was investigated.Specifically, control mice and Clostridium-inoculated mice weresensitized with oxazolone, and subsequently the inside of the rectumsthereof were treated with a 1% oxazolone/50% ethanol solution. Then,body weight loss was observed and measured. In addition, the colons weredissected, and analyzed histologically by HE staining.

The results show that the colitis proceeded along with persistent bodyweight loss in the control mice. Meanwhile, the body weight loss of theClostridium-abundant mice was reduced. In addition, it was also revealedthat portions having histological diseases such as mucosal erosion,edema, cellular infiltration, and hemorrhage were reduced in the colonof the Clostridium-abundant mice.

Example 16

Next, the influence, on the systemic immune response (systemic IgEproduction), of the colonization of a large number of Clostridium andthe resultant proliferation of Treg cells was investigated.Specifically, as described above, control mice andClostridium-inoculated mice were immunized by administeringalum-absorbed ovalbumin (OVA) twice at a 2-week interval. Then, serawere collected from these mice, and the OVA-specific IgE level thereofwas investigated by ELISA. In addition, splenic cells were collectedfrom the mice in each group, and IL-4 and IL-10 production by in-vitroOVA restimulation was investigated.

Results show that the IgE level was significantly lower in theClostridium-abundant mice than in the control mice. Moreover, the IL-4production by the OVA restimulation was reduced and the IL-10 productionthereby was increased in the splenic cells of the Clostridium-abundantmice sensitized with OVA and alum, in comparison with those of thecontrol mice.

Accordingly, in consideration of the results shown in Example 15 incombination, the induction of Treg cells by Clostridium in the colonplays an important role in local and systemic immune responses.

Example 17

Next, GF Balb/c were colonized with three strains of Clostridiumbelonging to cluster IV (strains 22, 23 and 32). Three weeks later,colonic Foxp3⁺ Treg cells were analyzed by FACS. Results show thatgnotobiotic mice colonized with three strains of Clostridium showed anintermediate pattern of Treg induction between GF mice and miceinoculated with all 46 strains.

Example 18

Next, it was investigated whether or not a spore-forming (for example, achloroform resistant) fraction of a fecal sample obtained from humanshad the effect of inducing proliferation or accumulation of regulatory Tcells similar to the spore-forming fraction of the fecal sample obtainedfrom mice.

Human stool from a healthy volunteer (Japanese, male, 29 years old) wassuspended with phosphate-buffered saline (PBS), mixed with chloroform(final concentration 3%), and then incubated in a shaking water bath for60 min. After evaporation of chloroform by bubbling with N₂ gas, thealiquots containing chloroform-resistant (for example, spore-forming)fraction of human intestinal bacteria were orally inoculated intogerm-free (GF) mice (IQI, 8 weeks old). The treated mice were kept in avinyl isolator for 3 weeks. The colon was collected and openedlongitudinally, washed to remove fecal content, and shaken in Hanks'balanced salt solution (HBSS) containing 5 mM EDTA for 20 min at 37° C.After removing epithelial cells and fat tissue, the colon was cut intosmall pieces and incubated with RPMI1640 containing 4% fetal bovineserum, 1 mg/ml collagenase D, 0.5 mg/ml dispase and 40 μg/ml DNase I(all manufactured by Roche Diagnostics) for 1 hour at 37° C. in ashaking water bath. The digested tissue was washed with HBSS containing5 mM EDTA, resuspended in 5 ml of 40% Percoll (manufactured by GEHealthcare) and overlaid on 2.5 ml of 80% Percoll in a 15-ml Falcontube. Percoll gradient separation was performed by centrifugation at 780g for 20 min at 25° C. The interface cells were collected and suspendedin staining buffer containing PBS, 2% FBS, 2 mM EDTA and 0.09% NaN₃ andstained for surface CD4 with Phycoerythrin-labeled anti-CD4 Ab (RM4-5,manufactured by BD Biosciences). Intracellular staining of Foxp3 wasperformed using the Alexa647-labeled anti-Foxp3 Ab (FJK-16s,manufactured by eBioscience) and Foxp3 Staining Buffer Set (manufacturedby eBioscience). The percentage of Foxp3 positive cells within the CD4positive lymphocyte population was analyzed by flow cytometry.

Results show that when the spore-forming (for example, the chloroformresistant) fraction of human intestinal bacteria was colonized in GFmice, the accumulation of Foxp3+ regulatory (Treg) cells in the coloniclamina propria of the mice was induced.

Next, it was investigated what species of bacteria grew by gavaging withchloroform-treated human stool.

Specifically, using a QIAamp DNA Stool mini kit (manufactured byQIAGEN), bacterial genomic DNA was isolated from the human stool from ahealthy volunteer as described above (human stool) or fecal pellets fromGF mice gavaged with chloroform-treated human stool (GF+Chloro.).Quantitative PCR analysis was carried out using a LightCycler 480(manufactured by Roche). Relative quantity was calculated by the ΔCtmethod and normalized to the amount of total bacteria, dilution, andweight of the sample. The following primer sets were used:

total bacteria (SEQ ID NO: 62) 5′-GGTGAATACGTTCCCGG-3′ and(SEQ ID NO: 63) 5′-TACGGCTACCTTGTTACGACTT-3′Clostridium cluster XIVa (Clostridium coccoides subgroup)(SEQ ID NO: 64) 5′-AAATGACGGTACCTGACTAA-3′ and (SEQ ID NO: 65)5′-CTTTGAGTTTCATTCTTGCGAA-3′ Clostridium cluster IV (Clostridium leptum)(SEQ ID NO: 66) 5′-GCACAAGCAGTGGAGT-3′ and (SEQ ID NO: 69)5′-CTTCCTCCGTTTTGTCAA-3′ Bacteroides (SEQ ID NO: 67)5′-GAGAGGAAGGTCCCCCAC-3′ and (SEQ ID NO: 68) 5′-CGCTACTTGGCTGGTTCAG-3′.

Results show that gavaged with chloroform-treated human stool had largeamounts of spore-forming bacteria, such as Clostridium clusters XIVa andIV, and a severe decrease of non-spore-forming bacteria, such asBacteroides, compared with the human stool before chloroform treatment.

Example 19

Human stool (2 g) from a healthy volunteer (Japanese, male, 29 y old)was suspended with 20 ml phosphate-buffered saline (PBS), mixed with orwithout chloroform (final concentration 3%), and incubated in a shakingwater bath for 60 min. The chloroform was then evaporated by bubblingwith N2 gas for 30 min. The suspensions of untreated human feces(designated as ‘huUT’) and chloroform-treated human feces (designated as‘huChloro’) were orally inoculated into Germ-free (GF) mice (IQI, 8 weekold) (250 μl/mouse). The suspension of huUT was inoculated into 4 GFmice, which were numbered from #A1 to #A4, and that of huChloro wasinoculated into 4 GF mice numbered from #B1 to #B4. Such GF mice whichwere inoculated with suspensions of feces or the like are also referredto as “ex-GF mice” hereinafter. Each group of ex-GF mice was separatelykept in a vinyl isolator to avoid further microbial contamination. After3 weeks, the small intestinal and colonic lamina propria lymphocytesfrom each mouse were separately collected, and examined for theexpressions of surface CD4 and intracellular Foxp3, Helios, IL-17 andIFN-γ by flow cytometry. For intracellular IL-17 and IFN-γ staining,isolated lymphocytes were stimulated in vitro with PMA and ionomycin for4 hours. Foxp3 is the transcription factor essential for thedifferentiation and function of Treg cells. Helios is a member of theIkaros transcription factor family and Helios− Foxp3+ Treg cells havebeen suggested to be Treg cells induced in the periphery [so calledinduced Treg (iTreg) cells]. As shown in FIGS. 1A-D, the percentages ofFoxp3+ Treg cells within CD4+ T cells in the small intestinal andcolonic lamina propria of both groups of ex-GF mice were increased,compared with those in GF mice. Marked increases were also observed forthe percentage of Helios-cells among Foxp3+ Treg cells in smallintestine and colon in both groups of ex-GF mice. Notably, besidesFoxp3+ Treg cells, a significant accumulation of IL-17-expressing CD4+cells (namely, Th17 cells) was observed in exGF+huUT mice, whereas itwas only marginally observed in exGF+huChloro mice (FIGS. 1E, F). Inboth groups of mice, the percentages of IFN-γ+ cells in CD4+ cells wereunchanged (FIGS. 1E, G).

Example 20

To investigate whether dead bacteria also have an effect on theinduction of Treg cells, the suspension of chloroform-treated humanfeces was autoclaved (121° C. for 20 min) and orally inoculated into GFmice (once a week for 4 weeks). After 4 weeks, mice were sacrificed, andthe colonic lamina propria lymphocytes from each mouse were examined forthe expression of CD4, Foxp3 and Helios by flow cytometry. As shown inFIG. 2, the inoculation of dead bacteria exhibited no effect on thenumbers of Foxp3+ cells or Helios-Foxp3+ cells. These results do notrule out the possibility that the amount of dead bacteria inoculated wasnot sufficient, but suggest that live bacteria are required for theinduction of Treg cells.

Example 21

To confirm the induction of Treg cells by chloroform-resistant bacteria,another stool was obtained from the same person on a different day,treated with chloroform, and inoculated into IQI GF mice (7 mice,numbered from #C1 to C7). After 3-4 weeks, mice from #C1 to #C5 weresacrificed, and the small intestinal and colonic lamina proprialymphocytes from each mouse were separately collected, and examined forthe expression of CD4 and Foxp3 by flow cytometry. Consistent with thefindings in Example 19, colonization with chloroform-treated human fecessignificantly induced the accumulation of Foxp3+CD4+ Treg cells incolonic and small intestinal lamina propria (FIG. 3). These resultsfurther support the notion that chloroform-resistant spore-formingbacteria can induce differentiation, proliferation and/or recruitment ofTreg cells in intestinal lamina propria.

Example 22

To test whether Treg cell induction by chloroform-resistantspore-forming fraction of human intestinal bacteria is horizontallytransmissible, IQI GF mice (6 mice, numbered from #D1 to #D6) werecohoused for 4 weeks with mice #C6 and #C7 in the same cage in a vinylisolator. Lamina propria lymphocytes from colon and small intestine wereisolated and examined for CD4 and Foxp3. Cohoused mice exhibited asignificant increase in the percentage of Foxp3+ cells among CD4+ cells(FIG. 4). Therefore, Treg cell induction by human intestinal bacteria ishorizontally transmissible. These results let us assume a role ofprominent components of the intestinal microbiota, rather than minorcomponents, for the induction of Treg cells.

Example 23

The frozen stock of cecal content from mouse #C4 was thawed, suspendedin 10 times its volume (w/v) of PBS, and passed through a 70 μm cellstrainer. The suspension was then treated with 3% chloroform, diluted2000- or 20000-fold with PBS, and orally inoculated into GF IQI mice(2.5×10⁵ or 2.5×10⁴ bacterial cells/250 μl/head, respectively). The2000-fold diluted sample was orally inoculated into 4 mice (designatedas exGF+2000, numbered from #E1 to #E4), whereas 20000-fold dilutedsample was inoculated into 8 mice (designated as exGF+20000, numberedfrom #F1 to #F8). After 3 weeks, the intestinal lamina proprialymphocytes were isolated and examined for CD4, Foxp3 and Helios. Both2000- and 20000-fold diluted samples similarly induced a markedaccumulation of Foxp3+CD4+ cells in the intestinal lamina propria (FIG.5). Therefore, the dose of bacteria for oral inoculation can beminimized to less than 2.5×10⁴ bacterial cells.

Example 24

The frozen stock of cecal content from mouse #F3, #F7 or #F8 wassuspended in 10 times its volume (w/v) of PBS, passed through a 70 μmcell strainer, and treated with 3% chloroform. Then, the fecalsuspension from mouse #F3 was orally inoculated into 5 GF mice (numberedfrom #G1 to #G5), that from #F7 mouse into 4 GF mice (numbered from #H1to #H4), and that from #F8 mouse into 4 GF mice (numbered from #I1 to#I4). After 4 weeks, lymphocytes from colonic and small intestinallamina propria were isolated and examined for CD4, Foxp3 and Heliosexpression by flow cytometry. All #F, #G, and #H mice exhibited asignificant increase in the percentage of Foxp3⁺ cells among CD4⁺ cellsin the intestinal lamina propria compared with untreated GF mice (FIG.6). Therefore, the Treg cell induction by human intestinal bacteriacolonizing in exGF+20000 mice is also transmissible. Moreover, as shownin the later meta 16S rDNA sequencing data (FIG. 8), these mice commonlyhad bacteria having 16S rDNA sequence similarities with 16S rDNAsequence similarities with 20 species of known bacteria (C. aminophilum,H. saccgarovorans, E. fissicatena, H. filiformis, C. clostridioforme, C.indolis, C. bolteae, Bacteroides sp. MANG, L. bacterium DJF_VP30,Ruminococcus sp. ID8, C. lavalense, C. symbiosum, E. contortum, C.saccharogumia, C. ramosum, F. plautii, C. scindens, Clostridium sp.2335, Clostridium sp. 14616 and cf Clostridium sp. MLG055).

Example 25

A frozen stock of the cecal content from #F8 mouse was serially dilutedwith 0.85% NaCl under an aerobic condition and plated onto BL agar.After culture at 37° C. for 2 or 4 days, 50 single colonies wereobserved. Of the 50 colonies, 29 were picked up, cultured for additional2 or 4 days at 37° C. by ABCM broth, and stored in EG stock medium (10%DMSO) at −80° C. The genomic DNA from each colony was isolated, and 16SrRNA coding gene sequence was analyzed. The sequence of 16S rRNA of eachcolony revealed that the 29 colonies observed were represented by threestrains, each having 100% similarity with Clostridium ramosum, 99.75%with Clostridium saccharogumia, 100% with Flavonifractor plautii, 99.17%with Clostridium hathewayi, 99.23% with Clostridium scindens, or 99.66%with Clostridium sp. 2335. Within the 29 colonies that were selectedfrom the original 50 colonies, only Clostridium saccharogumia,Clostridium ramosum, and Flavonifractor plautii were present (25, 3, and1 colonies, respectively). These 3 isolated strains were propagated,mixed and inoculated into GF IQI mice (4 mice, numbered from #J1 to J4).After 3-4 weeks, the colonic lamina propria lymphocytes were collected,and examined for the expressions of CD4, Foxp3, and Helios by flowcytometry. Foxp3+ cells or Helios-cells were not induced or only weaklyinduced by the colonization of these strains of bacteria in the colon(FIG. 7). These results suggest that the combination of Clostridiumsaccharogumia and Clostridium ramosum (both within cluster XVIII) wereinsufficient to induce Treg cells in the colon of mice. The effects ofFlavonifractor plautii were not clear, since the strain was onlyrepresented by 1 of the 29 colonies that were selected.

Example 26

The frozen glycerol stock of cecal content from #G2 mouse was suspendedwith PBS, seeded onto BL agar plate, and incubated for 48 hours,similarly to the procedure done in Example 19. Different from Example19, all bacteria on the plate were collected by scraping with a platescraper, suspended in TS broth and inoculated into GF IQI mice (4 mice,numbering from #K1 to #K4). It should be noted that the bacterialsuspension used in this experiment included bacteria that did notpropagate but survived on the plate. After 4 weeks, lamina proprialymphocytes from colon and small intestine of K1˜K4 mice were isolatedand examined for CD4, Foxp3 and Helios expression. All 4 mice exhibiteda significant increase in the percentages of Foxp3⁺ cells among CD4⁺cells (FIGS. 9A, 9B) and Helios⁻ cells among Foxp3⁺ Treg cells (FIGS.9A, 9C) in the intestinal lamina propria compared with untreated GFmice. Considering that the inoculation of mice with 6 strains ofbacteria propagated on the BL agar plate failed to induce Treg cells,bacteria that did not propagate but survived on the plate might beresponsible for the induction of Treg cells.

Example 27

Bacterial DNA was extracted from the cecal contents of mouse #A1, #C4,#F8, #G2, #H3, #I3, #J3 and #K3. Variable region 1-2 (V1-2) in bacterial16S rRNA coding gene were amplified by PCR and used as template formetasequencing. Resulting sequences (3400 reads for each sample) wereclassified into operational taxonomic units (OTUs) on the basis ofsequence similarity (>97% identity). Representative sequences from eachOTU were compared with sequences in nucleic acid databases using BLASTto determine their closest relatives in known species. The numbers ofdetected reads and the closest relatives for each OTU are shown inTable 1. The relative abundances of OTUs having the same closestrelative in each cecal sample are shown in FIG. 8. In mouse #A1, 153OTUs (their closest relatives were 93 species) were identified and halfof them were related to Bacteroides species. In contrast, in mouse #C4,113 OTUs were identified and most of them were related to speciesbelonging to the family Clostridiaceae. In mouse #F8, #G2, #H3, #I3, #J3and #K3, 97-68 OTUs were identified. In these mice, in which Treg cellaccumulation was observed in the intestine, the majority of bacteriaconsisted of bacteria having 16S rDNA sequence similarities with C.aminophilum, H. saccgarovorans, E. fissicatena, H. filiformis, C.clostridioforme, C. indolis, C. bolteae, Bacteroides sp. MANG, L.bacterium DJF_VP30, Ruminococcus sp. ID8, C. lavalense, C. symbiosum, E.contortum, C. saccharogumia, C. ramosum, F. plautii, C. scindens,Clostridium sp. 2335, Clostridium sp. 14616 and cf Clostridium sp.MLG055.

In mouse #J3, in which Treg accumulation was not observed, 3 OTUs weredetected. Each has the 16S rDNA sequence similarity with C.saccharogumia, C. ramosum or F. plautii. These results suggest that thecombination of these three species are insufficient to induce theintestinal Treg cells accumulation.

Example 28

Bacterial strains were isolated from the cecal contents of mouse #F8,#G2, #I1 and #K3 using BL agar or EG agar plates. Applicant picked-up144 colonies from EG agar plates and 116 colonies from BL agar plates.BLAST search of 16S rRNA coding sequence of these clones revealed thatthey belonged to 17 species, and each had 93-100% similarities with C.indolis, C. bolteae, Bacteroides sp. MANG, L. bacterium DJF_VP30, A.colihominis, Ruminococcus sp. ID8, C. lavalense, C. symbiosum, E.contortum, C. saccharogumia, C. ramosum, F. plautii, C. hathewayi, C.scindens, Clostridium sp. 2335, Clostridium sp. 14616 and cf Clostridiumsp. MLG055) (Table 2). They all belonged to Clostridium clusters IV,XIVa or XVIII (2 species of cluster IV, 12 of cluster XIVa, 1 of clusterXVI and 2 of cluster XVIII).

Example 29

Of the colonies selected in Example 28, additional colonies were pickedand isolated and these strains were cultured using EG and BL media.BLAST search of 16S rRNA coding sequence of these clones revealed thatthey belonged to a total of 31 species (including the species mentionedin Example 28), and each had 93-100% similarities with Clostridiumsaccharogumia, Clostridium ramosum JCM1298, Clostridium ramosum,Flavonifractor plautii, Pseudoflavonifractor capillosus ATCC 29799,Clostridium hathewayi, Clostridium saccharolyticum WM1, Bacteroides sp.MANG, Clostridium saccharolyticum, Clostridium scindens, Lachnospiraceaebacterium 5_1_57FAA, Lachnospiraceae bacterium 6_1_63 FAA, Clostridiumsp. 14616, Clostridium bolteae ATCC BAA-613, cf. Clostridium sp. MLG055,Erysipelotrichaceae bacterium 2_2_44A, Clostridium indolis, Anaerostipescaccae, Clostridium bolteae, Lachnospiraceae bacterium DJF_VP30,Lachnospiraceae bacterium 3_1_57FAA_CT1, Anaerotruncus colihominis,Anaerotruncus colihominis DSM 17241, Ruminococcus sp. ID8,Lachnospiraceae bacterium 2_1_46FAA, Clostridium lavalense, Clostridiumasparagiforme DSM 15981, Clostridium symbiosum, Clostridium symbiosumWAL-14163, Eubacterium contortum, Clostridium sp. D5, Oscillospiraceaebacterium NML 061048, Oscillibacter valericigenes, Lachnospiraceaebacterium A4, Clostridium sp. 316002/08, and Clostridiales bacterium1_7_47FAA, Blautia cocoides, Anaerostipes caccae DSM 14662 (Table 3).The stocks of bacterial strains were stored in 10% glycerol stock plusthe media used to grow the cultures, and tubes were stored in a −80° C.freezer.

Example 30

To investigate whether the strains in Example 29 have the ability toinduce Tregs in GF mice, 31 strains on Table 3 were mixed at equalamounts of media volume using TS media and inoculated into GF mice. Adetailed analysis of the 16S rRNA sequences revealed that 8 of the 31strains overlapped with other strains (see Table 3, indicated by anasterisk), resulting in 23 distinct bacterial strains. As shown in FIG.10, when orally administered to GF mice, the mixture of the 23 strains(23mix) induced very strong levels of Tregs (35-40% in the colon laminapropria, >10% in the small intestine; FIG. 10). These Tregs observedwith colonization by 23mix were mostly Helios⁻.

Example 31

To investigate whether the abundant members of the intestinal microbiotain the chloroform-resistant fraction of human intestinal bacteria,rather than the minor members, drive the induction of Treg cells, adultGF mice were inoculated with diluted caecal samples from mice that hadbeen inoculated with the chloroform-resistant fraction of humanintestinal bacteria (+huChlo mice) as described in example 19. As shownin FIG. 11, even when the huChlo mice cecal samples were diluted(diluted 2×10⁴ and 2×10⁵) to create +2×10⁴ mice and 2×10⁵ micerespectively, Tregs were induced in these adult GF mice.

Example 32

To investigate whether the mix of 23 strains in Example 30 has theability to induce Tregs in adult GF IQI mice more effectively thanFaecalibacterium prausnitzii, a well-known human Clostridia straincharacterized for enhancing regulatory cell functions, 23 strains intable 4 were mixed in equal amounts with media to make a cocktail, whichwas then administered to adult IQI GF mice. For comparison,Faecalibacterium prausnitzii was administered to another group of IQI GFmice. As shown in FIG. 12, when orally administered to adult IQI GFmice, the mixture of the 23 strains (23-mix) induced higher levels ofTregs than Faecalibacterium prausnitzii. Faecalibacterium prausnitzii(+Faecali.) showed negligible levels of Treg induction.

Example 33

To investigate whether the microbiota communities in the +2×104 mice,described in example 31, were stable, serial oral inoculation of adultGF mice was performed to create +2×10⁴-re mice (secondary inoculation)and +2×10⁴-re-re(tertiary inoculation). As shown in FIG. 13 there wassignificant induction of Tregs in both the +2×10⁴-re mice and the+2×10⁴-re-re mice. To further eliminate nonessential components of themicrobiota for Treg cell induction, the caecal content of +2×10⁴ mice,described in example 31, was again diluted 2×10⁴-fold and orallyinoculated into another set of adult GF mice (+(2×10⁴)² mice). As shownin FIG. 13, the +(2×10⁴)² mice exhibited a marked accumulation of Tregcells in the colon.

Example 34

To assess the composition of the gut microbiota in +huUT (+hu), +huChlo,+2×10⁴, +2×10⁴-re and +(2×10⁴)², described in example 19, example 31,and example 33, bacterial DNA was extracted from the caecal contents ofthese adult mice. The variable region (V1-V2) of the bacterial 16Sribosomal DNA (rDNA) was amplified and metasequencing using a 454sequencer was performed. The resulting sequences (3400 reads for eachsample) were classified into operational taxonomic units (OTUs) based onsequence similarity (>96% identity). Representative sequences from eachOTU were compared with sequences deposited in publicly available 16S andgenome databases using BLAST to determine their closest species. Asshown in FIG. 14, in +hu mice, OTUs belonging to Bacteroidetes accountedfor about 50% of the caecal microbial community. In contrast, in mostOTUs in +huChlo mouse were related to species belonging to Clostridia.In +2×10⁴, +2×10⁴-re and +(2×10⁴)² mice, the majority of bacteriaconsisted of bacteria having 16S rDNA sequence similarities with about20 species of Clostridia belonging to cluster XIVa (also referred to asC. leptum group), IV, XVI, and XVIII, listed in FIG. 14.

Example 35

A meta analysis of 16S rDNA of caecal contents from mice inoculated withthe 23 strains isolated in example 30 (+23-mix mice) confirmed thepresence of 17 of the 23 strains listed in FIG. 14 and Table 4. Todetermine whether these 17 strains could induce Treg cells, a mixture ofthese 17 strains was inoculated into adult GF mice (+17-mix mice), Eachbacterial strain was cultured in 2 mL EG liquid media and grown toconfluence, and then these starter cultures were mixed into a 50 mL tube(2 mL×17 strains=34 mL). The bacteria were spun down into a pellet andresuspended in 10 mL PBS. A 200 uL aliquot, containing ˜1×10⁶-1×10⁷ ofeach strain, was used to inoculate the adult GF mice. As shown in FIG.15, when orally administered to adult IQI, BALB, and B6 mice, themixture of 17 strains was able to induce Tregs in these three mousemodels.

Example 36

To investigate whether each of the 17 strains defined in example 35could individually induce Tregs, adult GF mice were monocolonized withone of each of the 17 strains. As shown in FIG. 16, adult GF micemonocolonized with a single strain exhibited low to intermediate levelsof Treg. Importantly, no single strain induced Tregs to the same extentas the mix of 17 strains.

Example 37

To investigate whether subsets of the 17 strains described in example 35could induce Tregs, randomly selected combinations of 3-5 strains weremade: 3-mix, 5mix A, 5-mix B, and 5-mix C, as shown in table 4, and usedto inoculate adult GF mice. As shown in FIG. 17, only the 5-speciesmixes induced significant increases in the frequency of Treg cells, themagnitude of which was intermediate compared with that observed in+17-mix mice.

Example 38

To investigate the benefits of administration of the mix of the 17strains described in example 35 (17-mix), adult SPF mice were orallyinoculated with either 17-mix or control media and assessed for theinduction of Foxp3+ Treg cells three weeks later. As shown in FIG. 18,there was a significant increase in the frequency of colonic Foxp3⁺ Treg(CD4) cells after three weeks of treatment.

Example 39

To evaluate the benefit of administration of 17-mix in an animal modelof allergic diarrhea, adult SPF mice were orally inoculated with 17-mixor control media while being treated with ovalbumin (OVA), an inducer ofallergic diarrhea. As shown in FIG. 19, the occurrence and severity ofdiarrhea (diarrhea score) was significantly reduced in mice fed 17-mixrelative to control mice.

Example 40

To evaluate the benefit of administration of 17-mix in an animal modelof colitis. Adult SPF mice were orally inoculated with either 17-mix orcontrol media while being treated with trinitrobenzene sulfonic acid(TNBS), a frequently used experimental inducer of colitis. As shown inFIG. 20, SPF 17-mix mice demonstrated lower mortality than control miceon exposure to TNBS.

Example 41

To evaluate the usefulness of the strains represented in 17-mix as adiagnostic and monitoring tool for ulcerative colitis, we examined therelative abundance of the 17 strains in healthy and ulcerative colitis(UC) human subjects using draft genomic sequences of the 17 strains andpublicly available human faecal microbiome genomes generated through theEuropean MetaHIT project. UC subjects (N=20) showed a reduction of the17 strains compared to healthy subjects (N=15), as shown in FIG. 21.

SEQ ID NOs.: OTU136; OTU46; OTU221; OTU9; OTU296; OTU21; OTU166; OTU73;OTU174; OTU14; OTU55; OTU337; OTU314; OTU195; OTU306; OTU87; OTU86;OTU152; OTU253; OTU259; OTU281; OTU288; OTU334; OTU359; OTU362; orOTU367 are SEQ ID NOs. 19-44, respectively.

INDUSTRIAL APPLICABILITY

As has been described above, the compositions and methods describedherein make it possible to provide an excellent and well-characterizedcomposition for inducing proliferation or accumulation of regulatory Tcells (Treg cells) by utilizing certain human-derived bacteria belongingto the Clostridia class or a physiologically active substance or thelike derived from the bacteria. Since the bacterial composition hasimmunosuppressive effects, the bacterial composition can be used, forexample, to prevent or treat autoimmune diseases or allergic diseases,as well as to suppress immunological rejection in organ transplantationor the like. In addition, healthy individuals can easily and routinelyingest the bacterial composition, such as in food or beverage, (e.g., ahealth food), to improve their immune functions.

TABLE 1A OTU The number of closest relative in known species name Closerelative #C4 #F8 #G2 #H3 #I3 #J3 218 bacterium ic337 9 0 0 0 0 0 104bacterium ic1395 4 0 0 0 0 0 60 64 249 Bacteroides uniformis 0 0 0 0 0 1 16 Bacteroides vulgatus 0 0 0 0 0 1 233 beta proteobacterium GMD 15D040 0 1 0 0 1 138 Bifidobacterium pseudocatenulatum 0 0 0 0 0 2 228butyrate-producing bacterium M104/1 0 0 1 0 0 3 31 73  cf. Clostridiumsp. MLG055 10 0 20 0 0 15 227 Clostridiaceae bacterium bSSV31 2 0 0 0 00 311 Clostridiaceae bacterium FH042 0 0 0 0 0 1 29 52 321Clostridiaceae bacterium NML 061030 3 0 3 0 0 8 33 156 Clostridiaceaebacterium SH021 10 0 0 0 0 0 183 Clostridiales bacterium DJF_B152 27 0 00 0 0 95 365 Clostridium citroniae 2 0 2 0 0 13 106 146  Clostridiumglycyrrhizinilyticum 2 1 0 0 0 0 91 105 102 178 Clostridium innocuum 320 0 0 0 0 203 292 318 69 80 325 Clostridium lactatifermentans 32 0 0 0 00 47 335 Clostridium methoxybenzovorans 13 0 0 0 0 0 147 175 298 344Clostridium sp. CE6 317 0 0 0 0 1 209 Clostridium sp. CYP2 1 0 0 0 0 0322 Clostridium sp. RT8 5 0 0 0 0 0 223 Clostridium sp. SH-C52 0 1 0 0 00  48 Clostridium xylanovorans 0 0 0 0 0 1 352 Desulfotomaculum sp. CYP11 0 0 0 0 0 132 154 283 Dorea longicatena 0 0 0 0 0 1 164 177 Eggerthella lenta 0 0 0 0 0 0 304 Escherichia coli 0 0 0 0 1 0 155Eubacterium dolichum 150 0 0 0 0 0  66 Eubacterium eligens 7 0 0 0 0 081 219 Eubacterium ramulus 36 0 0 0 0 0 287 Eubacterium siraeum 0 0 0 01 0  53 Eubacterium yurii 0 0 0 1 0 0 41 71 212 222 320 Faecalibacteriumprausnitzii 116 0 0 0 0 0 210 217 271 305 Faecalibacterium sp. DJF_VR20376 0 0 0 0 0 63 197 301 Firmicutes bacterium DJF_VP44 5 0 0 0 0 0 324Fusobacterium periodonticum 0 0 1 0 0 0 180 294  Gram-negative bacteriumcL10-2b-4 0 0 0 0 0 2 190 358  human intestinal bacterium julong 601 8 00 0 0 0 153 184 198 265 Lachnospiraceae bacterium 2 0 0 0 0 1 DJF_RP14171 Lactobacillus murinus 0 0 1 0 0 0  17 Odoribacter splanchnicus 13 00 0 0 0 267 Porphyromonas catoniae 0 0 1 0 0 0 145 Prevotellamelaninogenica 0 0 0 0 0 1  8 Prevotella nanceiensis 0 0 1 0 0 0 243Prevotellaceae bacterium DJF_RP17 0 1 0 0 0 0 103 Robinsoniellapeoriensis 1 0 0 0 0 4 98 127 Ruminococcus gnavus 286 0 0 0 0 0 43 99102 159 275 Ruminococcus sp. YE58 1 0 0 0 0 0 341 342 Ruminococcus sp.ZS2-15 53 0 0 0 0 0 252 Streptococcus australis 0 0 1 0 0 0 130 191 272Subdoligranulum sp. DJF_VR33k2 27 0 0 0 0 0 351 Subdoligranulumvariabile 1 0 0 0 0 0  56 unidentified bacterium ZF3 6 0 0 0 0 0 257 282284 327 Ruminococcus sp. K-1 322 0 0 0 0 0 329 332 87 124 204 259Eubacterium fissicatena 56 29 86 15 43 28 310 330 234 348  Eubacteriumcontortum 2 8 0 6 0 0  90 Lachnospiraceae bacterium A4 2 0 0 0 0 0 2 6182 92 111 Clostridium aminophilum 565 522 514 380 374 376 163 225 266288 312 336 355 359 367 281 296  Clostridium scindens 18 15 29 25 14 17224 254 264 Roseburia hominis 2 1 0 0 1 0 350 Ruminococcus sp. END-1 1 01 0 0 0 151 242 340 Hydrogenoanaerobacterium 141 205 199 138 175 140saccharovorans 44 101 110 119 131 Clostridium clostridioforme 12 20 2575 62 71 135 137 214 260 54 77 97 121 179 187 Clostridium symbiosum 3154 24 19 19 6 202 261 306 326 345 366 27 93 136 182 240 Clostridiumsaccharogumia 257 262 200 373 405 307 313 328 333 Bacteroides capillosus3 6 3 1 3 0 280 Holdemania filiformis 33 46 41 15 31 33 100 120 140 143166 Clostridium sp. 14616 97 165 493 287 287 153 194 229 237 276 297 307315 319 354 67 221 347 Flavonifractor plautii 12 17 34 25 30 29 85 107butyrate-producing bacterium T2-145 2 0 0 1 0 0 19 40 161 189 195 220Clostridium lavalense 75 285 278 475 298 197 238 262 269 303 334 45 94109 114 125 Ruminococcus sp. ID8 30 114 140 135 127 141 215 248 268 314337 Anaerotruncus colihominis 1 6 3 3 8 2 46 199 213 270 278 Clostridiumramosum 28 74 67 97 110 189 35 37 55 89 129 152 Lachnospiraceaebacterium 47 268 321 185 232 243 160 245 279 356 DJF_VP30 12 23 72 86174 Clostridium indolis 13 121 104 253 198 467 201 211 236 246 258 361364 4 9 13 14 22 28 38 Bacteroides sp. MANG 35 577 530 268 304 226 57 6276 78 144 186 231 241 362 3 7 15 20 21 24 39 Clostridium sp. 2335 57 5741 587 637 712 68 70 96 113 115 116 49 117 181 302 339 Clostridiumbolteae 0 13 0 30 32 0 74 126 208 251 285 Clostridium hathewayi 0 1 0 00 0 32 112 Clostridium sp. 14774 0 0 268 0 0 0 50 155 196 253Oscillibacter valericigenes 0 14 7 6 7 4 30 188 Ruminococcus sp. M-1 0 00 0 0 3 Total: 3400 3400 3400 3400 3400 3400

TABLE 1B OTU The closest relative in Similarity The number OTU nameknown species (%) #A1 #C4 #F8 #G2 #H3 #I3 #J3 3 Clostridium sp. 233598.46 1 0 13 18 10 13 8 9 Bacteroides sp. MANG 98.15 14 0 324 16 153 172159 14 Bacteroides sp. MANG 99.07 4 34 46 401 28 27 14 15 Clostridiumsp. 2335 96.9 0 0 8 2 0 2 1 21 Clostridium sp. 2335 99.69 19 53 325 322376 410 358 23 Clostridium indolis 97.25 0 0 0 0 3 1 2 38 Bacteroidessp. MANG 96.26 0 0 6 0 1 1 4 46 Clostridium ramosum 99.67 47 28 70 67 85101 188 49 Clostridium bolteae 95.98 1 0 7 0 17 28 0 55 Lachnospiraceaebacterium 95.53 12 45 120 289 72 85 106 DJF_VP30 57 Bacteroides sp. MANG96.27 3 0 93 0 27 38 20 86 Clostridium indolis 98.78 1 0 22 0 43 43 0 87Eubacterium fissicatena 99.69 1 40 11 39 4 8 0 89 Lachnospiraceaebacterium 95.18 1 0 4 0 0 2 0 DJF_VP30 92 Clostridium aminophilum 90.090 2 0 1 0 1 0 101 Clostridium clostridioforme 98.76 1 9 6 3 12 5 12 111Clostridium aminophilum 91.64 0 1 0 0 0 1 1 114 Ruminococcus sp. ID895.98 0 4 3 40 0 1 18 119 Clostridium clostridioforme 98.77 0 1 1 9 0 12 125 Ruminococcus sp. ID8 97.25 0 0 11 12 13 15 43 131 Clostridiumclostridioforme 97.23 1 0 1 3 2 1 9 136 Clostridium saccharogumia 97.0210 1 23 16 36 43 12 137 Clostridium clostridioforme 98.15 1 0 12 10 2851 47 144 Bacteroides sp. MANG 97.81 1 0 2 30 1 2 0 152 Lachnospiraceaebacterium 95.55 10 0 129 27 56 135 137 DJF_VP30 161 Clostridiumlavalense 96.3 0 0 1 1 0 4 0 163 Clostridium aminophilum 90.74 0 0 3 0 12 0 165 Oscillibacter valericigenes 90.15 0 9 0 7 0 1 1 166 Clostridiumsp. 14616 98.45 2 35 14 44 26 32 26 173 Clostridium sp. 2335 98.33 0 0 00 0 1 0 174 Clostridium indolis 100 0 13 98 103 205 152 465 181Clostridium bolteae 97.56 0 0 5 0 12 2 0 182 Clostridium saccharogumia94.37 0 2 1 0 3 1 1 189 Clostridium lavalense 94.12 0 0 0 0 0 1 0 195Clostridium lavalense 98.47 0 0 47 0 33 31 0 196 Oscillibactervalericigenes 91.64 1 4 2 0 0 1 1 199 Clostridium ramosum 98.05 0 0 0 05 9 0 202 Clostridium symbiosum 97.52 0 0 0 0 0 1 0 204 Eubacteriumfissicatena 96.62 0 14 4 30 0 16 15 211 Clostridium indolis 94.19 0 0 00 1 1 0 214 Clostridium clostridioforme 95.06 0 1 0 0 0 4 0 221Flavonifractor plautii 99.69 6 11 17 34 25 30 29 224 Roseburia hominis88.54 0 2 0 0 0 1 0 225 Clostridium aminophilum 90.8 0 13 10 8 7 2 1 237Clostridium sp. 14616 99.07 7 0 42 88 100 105 76 246 Clostridium indolis95.11 0 0 1 1 0 1 0 253 Oscillibacter valericigenes 92.81 9 0 12 0 6 5 2259 Eubacterium fissicatena 98.78 1 0 13 17 11 19 13 262 Clostridiumlavalense 98.77 0 16 26 215 25 45 117 268 Ruminococcus sp. ID8 97.82 0 036 0 4 100 41 269 Clostridium lavalense 97.27 0 0 1 0 2 2 0 277Clostridium sp. 2335 98.16 15 0 146 62 127 125 283 279 Lachnospiraceaebacterium 95.55 1 0 11 0 5 10 0 DJF_VP30 280 Holdemania filiformis 93.914 33 46 41 15 31 33 281 Clostridium scindens 99.69 0 11 6 22 15 11 10286 Clostridium sp. 2335 97.49 0 0 8 0 3 6 3 287 Eubacterium siraeum87.3 0 0 0 0 0 1 0 288 Clostridium aminophilum 91.33 10 537 394 480 283249 291 296 Clostridium scindens 99.69 0 7 9 7 10 3 7 297 Clostridiumsp. 14616 94.82 0 21 41 52 27 55 22 303 Clostridium lavalense 98.73 2 038 0 45 104 54 304 Escherichia coli 100 0 0 0 0 0 1 0 306 Clostridiumsymbiosum 99.38 0 28 50 22 1 17 6 307 Clostridium sp. 14616 94.39 1 3261 82 129 90 25 312 Clostridium aminophilum 91.69 0 0 0 0 0 1 0 313Clostridium saccharogumia 98.01 5 254 238 184 127 361 294 314Ruminococcus sp. ID8 97.53 0 24 12 88 6 11 39 319 Clostridium sp. 1461693.19 0 0 1 0 0 5 0 326 Clostridium symbiosum 91.67 0 0 0 0 0 1 0 328Bacteroides capillosus 92.9 1 3 4 3 1 2 0 333 Bacteroides capillosus93.23 0 0 2 0 0 1 0 334 Clostridium lavalense 95.37 0 59 50 62 11 111 26337 Anaerotruncus colihominis 99.38 2 1 6 3 3 8 2 339 Clostridiumbolteae 96.63 0 0 0 0 1 2 0 340 Hydrogenoanaerobacterium 87 37 141 205199 138 175 139 saccharovorans 353 Clostridium sp. 2335 96.63 7 3 59 8763 80 54 359 Clostridium aminophilum 90.46 1 7 11 18 4 7 8 362Bacteroides sp. MANG 98.14 3 0 100 79 55 64 29 367 Clostridiumaminophilum 90.43 2 0 101 3 17 111 75

TABLE 2 The Max Origin of corresponding Similarity Clostridiaceae mouseCultured Strain OTU The close relative (%) Cluster sample Media strain 1OTU136 Clostridium 99 XVIII #F8 BL saccharogumia strain 2 OTU46Clostridium ramosum 100 XVIII #F8, #G2, BL, EG #J3 strain 3 OTU221Flavonifractor plautii 100 IV #F8, #G2 BL strain 4 OTU9 Clostridiumhathewayi 99 XIVa #F8, #G2 BL strain 5 OTU296 Clostridium scindens 99XIVa #F8 BL strain 6 OTU21 Clostridium sp. 2335 99 XIVa #F8, #G2 BLstrain 7 OTU166 Clostridium sp. 14616 99 XIVa #G2 BL OTU237 strain 8OTU73 cf. Clostridium sp. 99 XVI #G2 BL MLG055 strain 9 OTU174Clostridium indolis 99 XIVa #G2, #J3 EG strain 10 OTU166 Clostridium sp.14616 97 XIVa #I1 EG OTU181 Clostridium bolteae 98 strain 11 OTU14Bacteroides sp. MANG 99 XIVa #I1 EG strain 12 OTU55 Lachnospiraceae 96XIVa #I1 EG bacterium DJF_VP30 strain 13 OTU337 Anaerotruncus 99 IV #I1EG colihominis strain 14 OTU314 Ruminococcus sp. ID8 99 XIVa #I1 EGstrain 15 OTU195 Clostridium lavalense 99 XIVa #I1 EG strain 16 OTU306Clostridium symbiosum 99 XIVa #I1 EG strain 17 OTU87 Eubacteriumcontortum 99 XIVa #I1 EG

TABLE 3 Sequenced Similarity to Strain OTU length (bp) Closest StrainSimilarity BLAST other strains Strain 1 136 1179 Clostridiumsaccharogunia 99.75 RDPiso Clostridium ramosum JCM1298 96.78 genomeDStrain 2 46 1184 Clostridium ramosum 100 RDPiso Clostridium ramosumJCM1298 100 genomeD Strain 18 46 492 Clostridium ramosum 100 DDBJ Strain2 Clostridium ramosum 100 genomeDB (>99%) Strain 3 221 1152Flavonifractor plautii 100 RDPiso Pseudoflavonifractor capillosus ATCC29799 97.22 genomeD Strain 4 9 1154 Clostridium hathewayi 99.31 RDPisoClostridium saccharolyticum WM1 95.06 genomeD Strain 11 14 487Bacteroides sp. MANG 99.33 RDPiso Strain 4 Clostridium saccharolyticumWM1 94.9 genomeDB (>99%) Strain 19 9 474 Bacteroides sp. MANG 99 DDBJStrain 4 Clostridium saccharolyticum 94.96 genomeDB (>99%) Strain 20 14470 Bacteroides sp. MANG 99 DDBJ Strain 4 Clostridium saccharolyticum95.81 genomeDB (>99%) Strain 30 362 478 Bacteroides sp. MANG 99 DDBJStrain 4 Clostridium saccharolyticum 94.68 genomeDB (>99%) Strain 5 2961182 Clostridium scindens 99.23 RDPiso Lachnospiraceae bacterium5_1_57FAA 99.05 genomeD Strain 6 21 1203 Blautia coccoides 99.92 RDPisoLachnospiraceae bacterium 6_1_63FAA 96.43 genomeD Strain 7 166 1149Clostridium sp. 14616 99.56 RDPiso Clostridium bolteae ATCC BAA-61399.56 genomeD Strain 8 73 1199 cf. Clostridium sp. MLG055 99.42 RDPisoErysipelotrichaceae bacterium 2_2_44A 92.71 genomeD Strain 9 174 1189Clostridium indolis 99.24 RDPiso Anaerostripes caccae DSM 14662 97.73genomeD Strain 22 86 478 Clostridium indolis 100 DDBJ Strain 9Anaerostripes caccae 96.96 genomeDB (>99%) Strain 10 166 491 Clostridiumbolteae 98.03 RDPiso Clostridium bolteae ATCC BAA-613 97.15 genomeDStrain 12 55 487 Lachnospiraceae bacterium DJF_VP30 96.08 RDPisoLachnospiraceae bacterium 3_1_57FAA_CT1 99.12 genomeD Strain 13 337 490Anaerotruncus colihominis 100 RDPiso Anaerotruncus colihominis DSM 17241100 genomeD Strain 14 314 487 Ruminococcus sp. ID8 99.54 RDPisoLachnospiraceae bacterium 2_1_46FAA 96.5 genomeD Strain 15 195 488Clostridium lavalense 99.56 RDPiso Clostridium asparagiforme DSM 15981100 genomeD Strain 16 306 470 Clostridium symbiosum 99.78 RDPisoClostridium symbiosum WAL-14163 99.56 genomeD Strain 17 87 474Eubacterium contortum 99.34 RDPiso Clostridium sp. D5 99.12 genomeDStrain 21 87 490 Eubacterium contortum 99 DDBJ Strain 17 Clostridium sp.D5 99.13 genomeDB (>99%) Strain 23 152 491 Lachnospiraceae bacteriumDJF_VP30 95 DDBJ Lachnospiraceae bacterium 3_1_57FAA_CT1 98.48 genomeDStrain 24 253 476 Oscillospiraceae bacterium NML 061048 93 DDBJOscillibacter valericigenes 93.23 genomeD Strain 25 259 491 Eubacteriumcontortum 99 DDBJ Clostridium sp. D5 99.78 genomeD Strain 26 281 490Clostridium scindens 97 DDBJ Lachnospiraceae bacterium 5_1_57FAA 98.03genomeD Strain 27 288 488 Lachnospiraceae bacteriumA4 95 DDBJLachnospiraceae bacterium 3_1_57FAA_CT1 97.45 genomeD Strain 28 334 490Clostridium sp. 316002/08 98 DDBJ Clostridiales bacterium 1_7_47FAA99.56 genomeD Strain 29 359 488 Lachnospiraceae bacteriumA4 95 DDBJLachnospiraceae bacterium 3_1_57FAA_CT1 97.8 genomeD Strain 31 367 489Lachnospiraceae bacteriumA4 95 DDBJ Strain 29 Lachnospiraceae bacterium3_1_57FAA_CT1 97.8 genomeDB (>99%)

TABLE 4 Similarity Sequenced with the Database Corresponding length of16S Clostridia closest used for Strain OTU rDNA (bp) Closest speciesCluster species (%) BLAST strain 1 OTU136 1418 Clostridium saccharogumiaXVIII 99.75 RDPiso Clostridium ramosum 96.78 genomeDB JCM 1298 strain 2OTU48 1184 Clostridium ramosum XVIII 100 RDPiso Clostridium ramosum 100genomeDB JCM1298 strain 3 OTU221 1427 Flavonifractor plautii IV 100RDPiso Pseudoflavonifractor 97.22 genomeDB capillosus ATCC 29799 strain4 OTU9 1430 Clostridium hathewayi XIVa 99.31 RDPiso Clostridium 95.06genomeDB saccharolyticum WM1 strain 5 OTU296 1433 Clostridium scindensXIVa 99.23 RDPiso Lachnospiraceae bacterium 99.05 genomeDB 5 1 57FAAstrain 6 OTU21 1428 Blautia coccoides XIVa 99.92 RDPiso Lachnospiraceaebacterium 96.43 genomeDB 6 1 63FAA strain 7 OTU166 1432 Clostridium sp.XIVa 99.56 RDPiso Clostridium bolteae 99.56 genomeDB ATCC BAA-613 strain8 OTU73 1433 cf. Clostridium sp. XVI 99.42 RDPiso MLG055 genomeDBErysipelotrichaceae 92.71 bacterium 2_2_44A strain 9 OTU174 1434Clostridium indolis XIVa 99.24 RDPiso Anaerostipes caccae 97.73 genomeDBDSM 14662 strain 10 OTU166 1431 Clostridium bolteae XIVa 98.03 RDPisoClostridium bolteae 97.15 genomeDB ATCC BAA-613 strain 11 OTU14 1430Bacteroides sp. MANG XIVa 99.33 RDPiso Clostridium saccharolyticum 94.9genomeDB WM1 strain 12 OTU55 1431 Lachnospiraceae bacterium XIVa 96.08RDPiso DJF_VP30 genomeDB Lachnospiraceae bacterium 99.12 3_1_57FAA_CT1strain 13 OTU337 1418 Anaerotruncus colihominis IV 100 RDPisoAnaerotruncus colihominis 100 genomeDB DSM 17241 strain 14 OTU314 1429Ruminococcus sp. ID8 XIVa 99.54 RDPiso Lachnospiraceae bacterium 96.5genomeDB 2_1_46 FAA strain 15 OUT195 1430 Clostridium lavalense XIVa99.56 RDPiso Clostridium asparagiforme 100 genomeDB DSM 15981 strain 16OTU306 1430 Clostridium symbiosum XIVa 99.78 RDPiso Clostridiumsymbiosum 99.56 genomeDB WAL-14163 strain 17 OTU87 474 Eubacteriumcontortum XIVa 99.34 RDPiso Clostridium sp. D5 99.12 genomeDB strain 18OTU46 1422 Clostridium ramosum XVIII 100 DDBJ Clostridium ramosum 100genomeDB strain 19 OTU9 474 Bacteroides sp. MANG XIVa 99 DDBJClostridium saccharolyticum 94.96 genomeDB strain 20 OTU14 1430Bacteroides sp. MANG XIVa 99 DDBJ Clostridium saccharolyticum 95.81genomeDB strain 21 OTU87 490 Eubacterium contortum XIVa 99 DDBJClostridium sp. D5 99.13 genomeDB strain 22 OTU86 1424 Clostridiumindolis XIVa 100 DDBJ Anaerostipes caccae 96.96 genomeDB strain 23OTU152 1430 Lachnospiraceae bacterium XIVa 95 DDBJ DJF_VP30 genomeDBLachnospiraceae bacterium 98.48 3_1_57FAA_CT1 strain 24 OTU253 1427Oscillospiraceae bacterium IV 93 DDBJ NML 061048 genomeDB Oscillibactervalericigenes 93.23 strain 25 OTU259 491 Eubacterium contortum XIVa 99DDBJ Clostridium sp. D5 99.78 genomeDB strain 26 OTU281 1433 Clostridiumscindens XIVa 97 DDBJ Lachnospiraceae bacterium 98.03 genomeDB 5_1_57FAAstrain 27 OTU288 1431 Lachnospiraceae bacterium XIVa 95 DDBJ A4 genomeDBLachnospiraceae bacterium 97.45 3_1_57FAA_CT1 strain 28 OTU344 1429Clostridium sp. 316002/08 XIVa 98 DDBJ Clostridiales bacterium 99.56genomeDB 1_7_47FAA strain 29 OTU359 1430 Lachnospiraceae bacterium XIVa95 DDBJ A4 genomeDB Lachnospiraceae bacterium 97.8 3_1_57FAA_CT1 strain30 OTU362 1430 Bacteroides sp. MANG XIVa 99 DDBJ Clostridiumsaccharolyticum 94.68 genomeDB strain 31 OTU367 1430 Lachnospiraceaebacterium XIVa 95 DDBJ A4 genomeDB Lachnospiraceae bacterium 97.83_1_57AA_CT1 Similarity to other Mix Strain strain 23-mix 17-mix 5-mix-A5-mix-B 5-mix-C 3-mix strain 1 X X X X strain 2 strain 18 X >99% strain3 X X X X strain 4 X X X strain 5 X strain 6 X X X strain 7 X X X strain8 X X X strain 9 X X X strain 10 X strain 11 strain 4 99% strain 12 Xstrain 13 X X X strain 14 X X X strain 15 X X X strain 16 X X X strain17 strain 22 >99% strain 18 X X strain 19 strain 4 >99% strain 20 strain4 >99% strain 21 X X strain 22 strain 9 >99% strain 23 X strain 24 Xstrain 25 X strain 26 X X X strain 27 X X X strain 28 X X X strain 29 XX X strain 30 strain 4 >99% strain 31 strain 29 >99%

OTU3 (SEQ ID NO.: 70) GATGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAGCGAAGCACTAAGACGGATTTC TTCGGATTGAAGTCTTTGTGACTGAGCGGCGGACGGTGAGTAACGCGTGGGTAACCTGCC TCATACAGGGGGATAACAGTTAGAAATGACTGCTAATACCGCATAAGCGCACAGGACCGC ATGGTCTGGTGTGAAAAACTCCGGTGGTATGAGATGGACCCGCGTCTGATTAGCTAGTTG GAGGGTAACGGCCCACCGAAGGCGACGATCAGTAGCCGGCCTGAGAGGGTGAACGGCCAC ATTGGGACTGAGACACGGCCCAG OTU9 (SEQ ID NO.: 22)GATGAACGCTGGCGGCGGTGCTTAACACATGCAAGTCGAGCGAAGCGGT TTCGAGTGAAGTTTTGGATGGAATTGAAATTGACTTAGCGGCGGACGGGTGAGTAACGCG TGGGTAACCTGCCTTACACTGGGGGATAACAGTTAGAAATGACTGCTAATACCGCATAAG CGCACAGGGCCGCATGGTCTGGTGCGAAAAACTCCGGTGGTGTAAGATGGACCCGCGTCT GATTAGGTAGTTGGTGGGGTAACGGCCCACCAAGCCGACGATCAGTAGCCGACCTGAGAG GGTGACCGGCCACATTGGGACTGAGACACGGCCCAA OTU14 (SEQ ID NO.: 28)GATGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAGCGAAGCGGTTT CAATGAAGTTTTCGGATGGAATTGAAATTGACTTAGCGGCGGACGGGTGAGTAACGCGTG GGTAACCTGCCTTACACTGGGGGATAACAGTTAGAAATGACTGCTAATACCGCATAAGCG CACAGGGCCGCATGGTCTGGTGTGAAAAACTCCGGTGGTGTAAGATGGACCCGCGTCTGA TTAGGTAGTTGGTGGGGTAACGGCCACCAAGCCGACGATCAGTAGCCGACCTGAGAGGGT GACCGGCCACATTGGGGACTGAGACACGGCCCA OTU15 (SEQ ID NO.: 71)GATGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAGCGAAGCATTAA GACAGATTTCTTCGGATTGAAGTCTTTGTGACTGAGCGGCGGACGGGTGAGTAACGCGTG GGTAACCTGCCTCATACAGGGGGATAACAGTTAGAAATGACTGCTAATACCGCATAAGCG CACAGGGCCGCATGGTCTGGTGTGAAAAACTCCGGTGGTGTAAGATGGACCCGCGTCTGA TTAGGTAGTTGGTGGGGTAACGGCCCACCAAGCCGACGATCAGTAGCCGACCTGAGAGGG TGACCGGCCACATTGGGACTGAGACACGGCCCAA OTU21 (SEQ ID NO.: 24)GATGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAGCGAAGCGCTAA GACAGATTTCTTCGGATTGAAGTCTTTGTGGCTGAGCGGCGGACGGGTGAGTAACGCGTG GGTAACCTGCCTCATACAGGGGGATAACAGTTAGAAATGACTGCTAATACCGCATAAGCG CACAGGACCGCATGGTCTGGTGTGAAAAACTCCGGTGGTATGAGATGGACCCGCGTCTGA TTAGCTAGTTGGAGGGGTAACGGCCCACCAAGGCGACGATCAGTAGCCGGCCTGAGAGGG TGAACGGCCACATTGGGACTGAGACACGGCCCA OTU23 (SEQ ID NO.: 72)GATGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAACGAAGCATTTT GGAAGGAAGTTTTCGGATGGAATTCCTTAATGACTGAGTGGCGGACGGGTGAGTAACGCG TGGGGAACCTCCCTACTACAGGGGAGTAACAGCTGGAACGGACTGCTAATACCGCATAAG CGCACAGAATCGCATGATTCGGTGTGAAAGCTCCGGCAGTATAGGATGGTCCCGCGTCTG ATTAGCTGGTTGGCGGGTAACGGCCCACCAAGGCGACGATCAGTAGCCGGCTTGAGAGAG TGGACGGCCACATTGGGACTGAGACACGGCCCAA OTU38 (SEQ ID NO.: 73)GATGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAGCGAAGCGGTTT CAATGAAGTTTTCGGATGGAATTGAAATTGACTTAGCGGCGGACGGGTGAGTAACGCGTG GGTAACCTGCCTCATACAGGGGGATAACAGTTAGAAATGACTGCTAATACCGCATAAGCG CACAGGACCGCATGGTCTGGTGTGAAAAACTCCGGTGGTATGAGATGGACCCGCGTCTGA TTAGCTAGTTGGAGGGGTAACGGCCCACCAAGGCGACGATCAGTAGCCGGCCTGAGAGGG TGAACGGCCACATTGGGACTGAGACACGGCCCAG OTU46 (SEQ ID NO.: 20)GATGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAACGCGAGCACTT GTGCTCGAGTGGCGAACGGGTGAGTAATACATAAGTAACCTGCCCTAGACAGGGGGATAA CTATTGGAAACGATAGCTAAGACCGCATAGGTACGGACACTGCATGGTGACCGTATTAAA GTGCCTCAAAGCACTGGTAGAGGATGGACTTATGGCGCATTAGCTGGTTGGCGGGGTAAC GGCCCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGACCGGCCACACTGGGACTG AGACACGGCC CAG OTU49(SEQ ID NO.: 74) GATGAACGCTGGCGGCGTGCCTAACACACGCAAGACGAACGAAGCAATTAAAATGAAGTT TTCGGATGGATTTTTGATTGACTGAGTGGCGGACGGGTGAGTAACGCGTG GATAACCTGCCTCACACTGGGGGGATAACAGTTAGAAATGACTGCTAATACCGCATAAGC GCACAGTACCGCATGGTACGTGTGAAAACTACCGGTGGTGTGAGATGGAGTCCCGCGTCT GATTAGCCAGTTGGCGGGGTAACGGCCCACCAAAGCGACGATCAGTAGCCGACCTGAGAG GGTGACCGGCCACATTGGGGACTGAGACACGGGCCCAA OTU55 (SEQ ID NO.: 29)GATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAACGAAGTTACAC GGAGGAAGTTTTCGGATGGAATCGGTATAACTTAGTGGCGGACGGGTGAGTAACGCGTGG GAAACCTGCCCTGTACCGGGGGATAACACTTAGAAATAGGTGCTAATACCGCATAAGCGC ACGGAACCGCATGGTTCCGTGTGAAAAACTACCGGTGGTACAGGATGGTCCCGCGTCTGA TTAGCCAGTTGGCAGGGTAACGGCCTACCAAAGCGACGATCAGTAGCCGGCCTGAGAGGG TGAACGGCCACATTGGGACTGAGACACAGCCCA OTU57 (SEQ ID NO.: 75)GATGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAGCGAAGCGGTTT CGATGAAGTTTTCGGATGGATTTGAAATCGACTTAGCGGCGGACGGGTGAGTAACGCGTG GGTAACCTGCCTTACACTGGGGGATAACAGTTAGAAATGACTGCTAATACCGCATAAGCG CACAGGGCCGCATGGTCTGGTGCGAAAAACTCCGGTGGTGTAAGATGGACCCGCGTCTGA TTAGCCAGTTGGCAGGGTAACGGCCTACCAAAGCGACGATCAGTAGCCGGCCTGAGAGGG TGAACGGCCACATTGGGACTGAGACACGGCCCAA OTU73 (SEQ ID NO: 26)GATGAACGCTGGCGGCATGCCTAATACATGCAAGTCGAACGAAGTGAAGA TAGCTTGCTATCGGAGCTTAGTGGCGAACGGGTGAGTAACACGTAGATAACCTGCCTGTA TGACCGGGATAACAGTTGGAAACGACTGCTAATACCGGATAGGCAGAGAGGAGGCATCTC TTCTCTGTTAAAGTTGGGATACAACGCAAACAGATGGATCTGCGGTGCATTAGCTAGTTG GTGAGGTAACGGCCCACCAAGGCGATGATGCATAGCCGGCCTGAGAGGGCGAACGGCCAC ATTGGGACTGAGACACGGCCCAA OTU86 (SEQ ID NO.: 35)GATGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAACGAAGCATTNT TGGAAGGAAGTTTCGGATGGAATTCCTTAATGACTGAGTGGCGGACGGGTGAGTAACGCG TGGGGAACCTACCCTATACAGGGGGATAACAGCTGGAAACGGCTGCTAATACCGCATAAG CGCACAGAATCGCATGATTCGGTGTGAAAAGCTCCGGCAGTATAGGATGGTCCCGCGTCT GATTAGCTGGTTGGCGGGTAACGGCCCACCAAGGCGACGATCAGTAGCCGGCTTGAGAGA GTGGACGGCCACATTGGGACTGAGACACGGCCCAA OTU87 (SEQ ID NO.: 34)GATGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAGCGAAGCGCTTT ACTTAGATTTCTTCGGATTGAAAGTTTTGCGACTGAGCGGCGGACGGGTGAGTAACGCGT GGGTAACCTGCCTCATACAGGGGGATAACAGTTAGAAATGACTGCTAATACCGCATAAGA CCACAGTACCGCATGGTACAGTGGGAAAAACTCCGGTGGTATGAGATGGACCCGCGTCTG ATTAGCTAGTTGGTAAGGTAACGGCTTACCAAGGCGACGATCAGTAGCCGACCTGAGAGG GTGACCGGCCACATTGGGACTGAGACACGGCCCA OTU89 (SEQ ID NO.: 76)GATGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAACGAAGCATTTT GGAAGGAAGTTTTCGGATGGAATCGGTATAACTTAGTGGCGGACGGGTGAGTAACGCGTG GGAAACCTGCCCTGTACCGGGGGATAACACTTAGAAATAGGTGCTAACACCGCATAAGCG CACGGAACCGCATGGTTCTGTGTGAAAAAACTCCGGTGGTACAGGATGGTCCCGCGTCTG ATTAGCCAGTTGGCGAGGGTAACGGCCTACCAAAGACGACGATCAGTAGCCGGCCTGAGA GGGTGAACGGCCACATTGGGACTGAGACACGGCCCAA OTU92 (SEQ ID NO.: 77)GATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAACGGAGTTATGC AGAGGAAGTTTTCGGATGGAATCGGCGTAACTTAGTGGCGGACGGGTGAGTAACGCGTGG GAAACCTGCCCTGTACCGGGGGATAACACTTAGAAATAGGTGCTAATACCGCATAAGCGC ACAGCTTCACATGAGGCAGTGTGAAAAACTCCGGTGGTGTAAGATGGACCCGCGTCTGAT TAGGTAGTTGGTGGGGTAACGGCCCACCAAGCCGACGATCAGTAGCCGACCTGAGAGGGT GACCGGCCACATTGGGACTGAGACACGGCCCA OTU101 (SEQ ID NO.: 78)GATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAACGAAGCAATTA AGATGAAGTTTTCGGATGGAATCTTGATTGACTGAGTGGCGGACGGGTGAGTAACGCGTG GATAACCTGCCTCACACTGGGGGATAACAGTTAGAAATGACTGCTAATACCGCATAAGCG CACAGTGCCGCATGGCAGTGTGTGAAAAACTCCGGTGGTGTGAGATGGATCCGCGTCTGA TTAGCCAGTTGGCGGGGTAACGGCCACCGAAAGCGACGATCAGTAGCCGACCTGAGAGGG TGACCGGCCACACTGGGACTGAGACACGGCCCA OTU111 (SEQ ID NO.: 79)GATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAACGAAGTTACAC AGAGGAAGTTTTCGGATGGAATCGGTATAACTTAGTGGCGGACGGGTGAGTAACGCGTGG GAAACCTGCCCTGTACCGGGGGATAACACTTAGAAATAGGTGCTAATACCGCATAAGCGC ACAGCTTCACATGAAGCAGTGTGAAAAACTCCGGTGGTACAGGATGGTCCCGCGTCTGAT TAGCTGGTTGGCGGGGTAACGGCCCACCAAGGCGACGATCAGTAGCCGGCTTGAGAGAGT GGACGGCCACATTGGGACTGAGACACGGCCCA OTU114 (SEQ ID NO.: 80)GATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAGCGAAGCGCTGT TTTCAGAATCTTCGGAGGAAGAGGACAGTGACTGAGCGGCGGACGGGTGAGTAACGCGTG GGCAACCTGCCTCATACAGGGGGATAACAGTTAGAAATGACTGCTAATACCGCATAAGCG CACAGGACCGCATGGTGTAGTGTGAAAAACTCCGGTGGTATGAGATGGACCCGCGTCTGA TTAGCCAGTTGGCAGGGTAACGGCCTACCAAAGCGACGATCAGTAGCCGGCCTGAGAGGG TGAACGGCCACATTGGGACTGAGACACGGCCCA OTU119 (SEQ ID NO.: 81)GATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAACGAAGCAATTA AGATGAAGTTTTCGGATGGAATCTTGATTGACTGAGTGGCGGACGGGTGAGTAACGCGTG GATAACCTGCCTCACACTGGGGGATAACAGTTAGAAATGACTGCTAATACCGCATAAGCG CACAGTGCCGCATGGCAGTGTGTGAAAAACTCCGGTGGTGTGAGATGGATCCGCGTCTGA TTAGCCAGTTGCGGGGTAACGGCCCGACCAAAGCGACGGATCAGTAGCCGACCTGAGAGG GTNACCGGCCACATTGGGACTGAGACACGGCCCA OTU125 (SEQ ID NO.: 82)GATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAGCGAAGCGCTGT TTTCAGAATCTTCGGAGGAAGAGGACAGTGACTGAGCGGCGGACGGGTGAGTAACGCGTG GGCAACCTGCCTCATACAGGGGGATAACAGTTAGAAATGACTGCTAATACCGCATAAGCG CACAGGACCGCATGGTGTAGTGTGAAAAACTCCGGTGGTATGAGATGGACCCGCGTCTGA TTAGGTAGTTGGTGGGTAAAGGCTACCGAAGCCGACGATCAGTAGCCGACCTGACGAGGG TGACCGGCCACGATTGGGACTGAGACACGGCCCAA OTU131 (SEQ ID NO.: 83)GATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAACGAAGCAATTA AGATGAAGTTTTCGGATGGAATCTTGATTGACTGAGTGGCGGACGGGTGAGTAACGCGTG GATAACCTGCCTCACACTGGGGGATAACAGTTAGAAATGACTGCTAATACCGCATAAGCG CACAGTGCCGCATGGCAGTGTGTGAAAAACTCCGGTGGTGTGAGATGGATCCGCGTCTGA TTAGCCAGTTGCGGGTAACGGCCACCGAAAGCGACGATCAGTAGCCGACCTGACGAGGGT NACCGGCACATTGGGACTGAGACACGGCCCAA OTU136 (SEQ ID NO.: 19)GATGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAACGCGAGCACTT GTGCTCGAGTGGCGAACGGGTGAGTAATACATAAGTAACCTGCCCTTTACAGGGGGATAA CTATTGGAAACGATAGCTAAGACCGCATAGGTAAAGATACCGCATGGTAAGTTTATTAAA AGTGCCAAGGCACTGGTAGAGGATGGACTTATGGCGCATTAGCTAGTTGGTGAGGTAACG GCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGACCGGCCACACTGGGACTGA GACACGGCCC AG OTU137(SEQ ID NO.: 84) GATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAACGAAGCAATTAAGATGAAGTT TTCGGATGGAATCTTGATTGACTGAGTGGCGGACGGGTGAGTAACGCGTG GATAACCTGCCTCACACTGGGGGGATAACAGTTAGAAATGACTGCTAATACCGCATAAGC GCACAGTGCCGCATGGCAGTGTGTGAAAAACTCCGGTGGTGTGAGATGGATCCGCGTCTG ATTAGGTAGTTGGTGGGGTAACGGCCCACCAAGCCGACGATCAGTAGCCGACCTGAGAGG GTGACCGGCCACATTGGGACTGAGACACGGCCCAA OTU144 (SEQ ID NO.: 85)GATGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAGCGAAGCGGTTT CGATGAAGTTTTTGGATGGAATTGAAATTGACTTAGCGGCGGACGGGTGAGTAACGCGTG GGTAACCTGCCTTACACTGGGGGATAACAGTTAGAAATGACTGCTAATACCGCATAAGCG CACAGGGCCGCATGGTCTGGTGCGAAAAACTCCGGTGGTGTAAGATGGACCCGCGTCTGA TTAGGTAGTTGGTGGGGTAACGGCCCACCGAAGCCGACGATCAGTAGCCGACCTGAGAGG GTGACCGGCACATTGGGACCTGAGACACGGGCCCA OTU152 (SEQ ID NO.: 36)GATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAACGAAGTTAGAC AGAGGAAGTTTTCGGATGGAATCGGTATAACTTAGTGGCGGACGGGTGAGTAACGCGTGG GAAACCTGCCCTGTACCGGGGGATAACACTTAGAAATAGGTGCTAATACCGCATAAGCGC ACGGAACCGCATGGGTTCTGTGTGAAAACTCCGGTGGTACAGGATGGTCCCGCGTCTGAT TAGCCAGTTGGCAGGGTAACGGCCTACCAAAGCGACGATCAGTAGCCGGCCTGAGAGGGT GAACGGCCACATTGGGACTGAGACACGGCCCAA OTU161 (SEQ ID NO.: 86)GATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAACGAAGCATTTT AGATGAAGTTTTCGGATGGATTCTGAGATGACTGAGTGGCGGACGGGTGAGTAACACGTG GATAACCTGCCTCACACTGGGGGACAACAGTTAGAAATGACTGCTAATACCGCATAAGCG CACAGTACCGCATGGTACGGTGTGAAAAACTCCGGTGGTACAGGATGGTCCCGCGTCTGA TTAGCCAGTTGGCAGGGTAACGGCCTACCAAAGCGACGATCAGTAGCCGGCCTGAGAGGG TGAACGGCCACATTGGGACTGAGACACGGCCCAA OTU163 (SEQ ID NO.: 87)GATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAACGAAGTTACAC GGAGGAAGTTTTCGGATGGAATCGGTATAACTTAGTGGCGGACGGGTGAGTAACGCGTGG GAAACCTGCCCTGTACCGGGGGATAACACTTAGAAATAGGTGCTAATACCGCATAAGCGC ACGGAACCGCATGGTTCCGTGTGAAAAACTCCGGTGGTACAGGATGGTCCCGCGTCTGAT TAGGTAGTTGGTGGGGTAACGGCCCACCAAGCCGACGATCAGTAGCCGACCTGAGAGGGT GACCGGCCACATTGGGACTGAGACACGGCCCA OTU165 (SEQ ID NO.: 88)GATGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAACGGAGCACCCT TGACTGAGGTTTCGGCCAAATGATAGGAATGCTTAGTGGCGGACTGGTGAGTAACGCGTG AGGAACCTACCTTCCAGAGGGGACGAACAGTTGGAACGACTGCTAATACCGCATGACGCA TGACCGGGGCGATCCCGGGCCGATGTCAAAGATTTTATTCGCTGGAAGATGGCCTCGCGT CTGATTAGCTAGATGGTGGGGTAACGGCCCACCATGGCGACGATCAGTAGCCGGACTGAG AGGTTGACCGGCCACATTGGGACTGAGATACGGCCCA OTU166 (SEQ ID NO.: 25)GATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAACGAAGCAATTA AAATGAAGTTTCGGATGGATTTTGATTGACTGAGTGGCGGACGGGTGAGTAACGCGTGGA TAACCTGCCTCACACTGGGGGATAACAGTTAGAAATGACTGCTAATACCGCATAAGCGCA CAGTACCGCATGGTACGGTGTGAAAAACTCCGGTGGTGTGAGATGGATCCGCGTCTGATT AGCCAGTTGGCGGGGTAACGGCCCACCAAAGCGACGATCAGTAGCCGACCTGAGAGGGTG ACCGGCCACGATTGGGACTGAGACACGGCCCA OTU173 (SEQ ID NO.: 123)GACGAACGCTGGCGGCGCGCCTAACACATGCAAGTCGAACGGAGTTGTGT TGAAAGCTTGCTGGATATACAACTTAGTGGCGGACGGGTGAGTAACGCGTGGGTAACCTG CCTCATACAGGGGGATAACAGTTAGAAATGACTGCTAATACCGCATAAGCGCACAGGATC GCATGGTCTGGTGTGAAAAACTCCGGTGGTATGAGATGGACCCGCGTCTGATTAACTAGT TGGAGGGGTAACGGCCCACCAAGGCGACGAGTCAGTAGCCGGCCTGAGAGGGTGAACGGC CACGATTGGGACTGAGACACGGCCCAG OTU174 (SEQ ID NO.: 27)GATGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAACGAAGCATTTT GGAAGGAAGTTTTCGGATGGAATTCCTTAATGACTGAGTGGCGGACGGGTGAGTAACGCG TGGGGAACCTGCCCTATACAGGGGGATAACAGCTGGAAACGGCTGCTAATACCGCATAAG CGCACAGAATCGCATGATTCGGTGTGAAAAGCTCCGGCAGTATAGGATGGTCCCGCGTCT GATTAGCTGGTTGGCGGGGTAACGGCCCACCAAGGCGACGATCAGTAGCCGGCTTGAGAG AGTGGACGGCCACATTGGGACTGAGACACGGCCCA OTU181 (SEQ ID NO.: 89)GATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAACGAAGCAATTT AAAATGAAGTTTTCGGATGGATTTTTGATTGACTGAGTGGCGGACGGGTGAGTAACGCGT GGATAACCTGCCTCACGACTGGGGGATAACAGTTAGAAATGACTGCTAATACCGCATAAG CGCACAGTACCGCATGGTACGGTGTGAAAAACTCCGGTGGTGTGAGATGGATCCGCGTCT GATTAGCCAGTTGCGGGGTAACGGCCCACCGAAAGCGACGATCAGTAGCCGACCTGAGAG GGTGACCGGCCACATTGGGGACTGAGACACGGCCCAA OTU182 (SEQ ID NO.: 90)GATGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAACGCGGGCAGCA ATGCCCGAGTGGCGAACGGGTGAGTAATACATAAGTAACCTGCCCTTTACAGGGGGATAA CTATTGGAAACGATAGCTAAGACCGCATAGGTAAAGATACCGCATGGTAAGTTTATTAAA AGTGCCAAGGCACTACGAGGGAGTAGTGATATGCGCATAGCTAGTTGGTGAGGTAACGGC TCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGACCGGCCACACTGGGACTGAGA CACGGCCCAG OTU189(SEQ ID NO.: 91) GATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAACGAAGCATTTTAGATGAAGTT TTCGGATGGATTCTGAGATGACTGAGTGGCGGACGGGTGAGTAACACGTG GATAACCTGCCTCACACTGGGGGACACAGTTAGAAATGACTGCTAATACCGCATAAGCGC ACAGCTTCACATGAAGCAGTGTGAAAAACTCCGGTGGTACAGGATGGTCCCGCGTCTGAT TAGCCAGTTGGCAGGGTAACGGCCTACCAAAGCGACGATCAGTAGCCGGCCTGAGAGGGT GAACGGCCACATTGGGACTGAGACACGGCCCAG OTU195 (SEQ ID NO.: 32)GATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAACGAAGCATTTT AGATGAAGTTTTCGGATGGATTCTGAGATGACTGAGTGGCGGACGGGTGAGTAACACGTG GATAACCTGCCTCACACTGGGGGACGAACAGTTAGAAATGACTGCTAATACCGCATAAGC GCACAGTACCGCATGGTACGGTGTGAAAAACTCCGGTGGTGTGAGATGGATCCGCGTCTG ATTAGCCAGTTGGCGGGTAACGGCCCACCGAAAGCGACGATCAGTAGCCGACCTGAGAGG GTGACCGGCCACATTGGGACTGAGACACGGCCCAA OTU196 (SEQ ID NO.: 92)GACGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAACGGAGCACCCC TGAATGAGGTTTCGGCCAAAGGAAGGGAATGCTTAGTGGCGGACTGGTGAGTAACGCGTG AGGAACCTGCCTTTCAGAGGGGACAACAGTTGGAAACGACTGCTAATACCGCATGACACA TGAATGGGGCATCCCATTGATGTCAAAGATTTATCGCTGAAAGATGGCCTCGCGTCCCAT TAGCTAGTAGGCGGGGTAACGGCCCACCTAGGCGACGATGGGTAGCCGGACTGAGAGGTT GACCGGCCACATTGGGACTGAGATACGGCCCA OTU199 (SEQ ID NO.: 93)GATGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAACGCGAGCACTT GTGCTCGAGTGGCGAACGGGTGAGTAATACATAAGTAACCTGCCCTAGACAGGGGGAGTA ACTATTGGAACGATAGCTAAGACCGCATAGGTACGGACACTGCGTGGTGACCGTATTAAA AGTAGCCTCAAAGACACTGGTAGAGGATGGACTTATGGCGCATTAGCTGGTTGGCGGGGT AACGGCCCACCCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGACCGGCCACACTGGG ACTGAGACAC GGCCCAGOTU202 (SEQ ID NO.: 94)GATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAACGAAGCAATTT AACGGAAGTTTTCGGATGGAAGTTGAATTGACTGAGTGGCGGACGGGTGAGTAACGCGTG GGTAACCTGCCTTGTACTGGGGGACAACAGTTAGAAATGACTGCTAATACCGCATAAGCG CACAGTATCGCATGATACAGTGTGAAAAACTCCGGTGGTACAAGATGGACCCGCGTCTGA TTAGCTAGTTGGAGGGGTAACGGCCCACCAAGGCGACGATCAGTAGCCGGCCTGAGAGGG TGAACGGCCACATTGGGACTGAGACACGGCCCAG OTU204 (SEQ ID NO.: 95)GATGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAGCGAAGCACTAA GACGGATTTCTTCGGATTGAAGTCTTTGTGACTGAGCGGCGGACGGGTGAGTAACGCGTG GGTAACCTGCCTCATACAGGGGGATAACAGTTAGAAATGACTGCTAATACCGCATAAGAC CACAGTACCGCATGGTACAGTGGGAAAAACTCCGGTGGTATGAGATGGACCCGCGTCTGA TTAGCTAGTTGGTAAGGTAACGGCTTACCAAGGCGACGATCAGTAGCCGACCTGAGAGGG TGACCGGCCACATTGGGACTGAGACACGGCCCA OTU211 (SEQ ID NO.: 96)GATGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAGCGAAGCGGTTT CGATGAAGTTTTCGGATGGATTTGAAATCGACTTAGCGGCGGACGGGTGAGTAACGCGTG GGTAACCTGCCTTACACTGGGGGATAACAGCTGGAAACGGCTGCTAATACCGCATAAGCG CACAGAATCGCATGATTCGGTGCGAAAAGCTCCGGCAGTATAGGATGGTCCCGCGTCTGA TTAGCTGGTTGGCGGGGTAACGGCCCACCAAGGCGACGATCAGTAGCCGGCTTGAGAGAG TGGACGGCCACATTGGGACTGAGACACGGCCCAA OTU214 (SEQ ID NO.: 97)GATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAACGAAGCAATTA AGATGAAGTTTTCGGATGGAATCTTGATTGACTGAGTGGCGGACGGGTGAGTAACGCGTG GGTAACCTGCCTCATACAGGGGGAGTAACAGTTAGAAATGACTGCTAATACCGCATAAGC GCACAGGGCTGCATGGCCTGGTGTGAAAAACTCCGGTGGTATGAGATGGACCCGCGTCTG ATTAGCTAGTTGGAGGGGTAACGGCCCACCAAGGCGACGATCAGTAGCCGGCCTGAGAGG GTGAACGGCCACATTGGGACTGAGACACGGCCCA OTU221 (SEQ ID NO.: 21)GATGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAACGGGGTGCTCA TGACGGAGGATTCGTCCAACGGATTGAGTTACCCAGTGGCGGACGGGTGAGTAACGCGTG AGGAACCTGCCTTGGAGAGGGGAATAACACTCCGAAAGGAGTGCTAATACCGCATGATGC AGTTGGGTCGCATGGCTCTGACTGCCAAAGATTTATCGCTCTGAGATGGCCTCGCGTCTG ATTAGCTAGTAGGCGGGGTAACGGCCCACCTAGGCGACGATCAGTAGCCGGACTGAGAGG TTGACCGGCCACATTGGGACTGAGACACGGCCCA OTU224 (SEQ ID NO.: 98)GATGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAGCGAAGCACCTT GGCGGATTTCTTCGGATTGAAGCCTTGGTGACTGAGCGGCGGACGGGTGAGTAACGCGTG GGTAACCTGCCCTGTACCGGGGGATAACACTTAGAAATAGGTGCTAATACCGCATAAGCG GCACACTTCACATGAAGCAGTGTGAAAAACTCCGGCGGTACAGGATGGTCCCGCGTCTGA TTAGCCAGTTGACAGGGTAACGGCCTACCAAAGCGACGATCAGTAGCCGGCCTGAGAGGG TGAACGGCCACATTGGGACTGAGACACGGCCCA OTU225 (SEQ ID NO.: 99)GATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAACGGAAGTTATG CAGAGGAAGTTTTCGGTATGGAATCGGCGTAACTTAGTGGCGGACGGGTGAGTAACGCGT GGGAAACCTGCCCTGTACCGGGGGAGTAACACTTAGAATAGGTGCTAATACCGCATAAGC GCACAGCTTCACATGAGGCAGTGTGAAAAACTCCGGTGGTACAGGATGGTCCCGCGTCTG ATTAGCCAGTTGGCAGGGTAACGGCCTACCAAAGCGACGATCAGTAGCCGGCCTGAGAGG GTGAACGGCCACATTGGGACTGAGACACGGCCCA OTU237 (SEQ ID NO.: 100)GATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAACGAAGCAATTG AAGGAAGTTTTCGGATGGAATTCGATTGACTGAGTGGCGGACGGGTGAGTAACGCGTGGA TAACCTGCCTCACACTGGGGGATAACAGTTAGAAATGACTGCTAATACCGCATAAGCGCA CAGTGCCGCATGGTACGGTGTGAAAAACTCCGGTGGTGTGAGATGGATCCGCGTCTGATT AGCCAGTTGGCGGGGTAACGGCCCACCAAAGCGACGATCAGTAGCCGACCTGAGAGGGTG ACCGGCCACATTGGGACTGAGACACGGCCCAA OTU246 (SEQ ID NO.: 101)GATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAACGGAGTTATGC AGAGGAAGTTTTCGGATGGAATCGGCGTAACTTAGTGGCGGACGGGTGAGTAACGCGTGG GAAACCTGCCCTATACAGGGGGATAACAGCTGGAAACGGCTGCTAATACCGCATAAGCGC ACAGAATCGCATGATTCGGTGTGAAAAGCTCCGGCAGTATAGGATGGTCCCGCGTCTGAT TAGCTGGTTGGCGGGGTAACGGCCCACCAAGGCGACGATCAGTAGCCGGCTTGAGAGAGT GGACGGCCACATTGGGACTGAGACACGGCCCAA OTU253 (SEQ ID NO.: 37)GACGAACGCTGGCGGCGTGCTTAACACATGCAAATCGAACGGAGCACCCT TGACTGAGGTTTCGGCCAAATGATAGGAATGCTTAGTGGCGGACTGGTGAGTAACGCGTG AGGAACCTGCCTTCCAGAGGGGGACAACAGTTGGAAACGACTGCTAATACCGCATGACGC ATGACCGGGGCATCCCGGGCATGTCAAAGATTTTATCGCTGGAAGATGGCCTCGCGTCTG ATTAGCTAGATGGTGGGGTAACGGCCCACCATGGCGACGATCAGTAGCCGGACTGAGAGG TTGACCGGCCACATTGGGACTGAGATACGGGCCCAG OTU259 (SEQ ID NO.: 38)GATGAACGCTGGCGGCGTGCTTAAACATGCAAGTCGAGCGAAGCGCTTTA CTTAGATTTCTTCGGATTGAAAAGTTTTGCGACTGAGCGGCGGACGGGTGAGTAACGCG TGGGTAACCTGCCTCATACAGGGGGATAACAGTTAGAAATGACTGCTAATACCGCATAAG ACCACGGTACCGCATGGTACAGTGGGAAAAACTCCGGTGGTATGAGATGGACCCGCGTCT GATTAGCTAGTTGGTAAGGTAACGGCTTACCAAGGCGACGATCAGTAGCCGACCTGAGAG GGTGACCGGCACATTGGGACCTGAGACACGGCCCAA OTU262 (SEQ ID NO.: 102)GATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAACGAAGCATTTT AGATGAAGTTTTCGGATGGATTCTGAGATGACTGAGTGGCGGACGGGTGAGTAACACGTG GATAACCTGCCTCACACTGGGGGACAACAGTTAGAAATGACTGCTAATACCGCATAAGCG CACAGTACCGCATGGTACAGTGTGAAAAACTCCGGTGGTGTGAGATGGATCCGCGTCTGA TTAGCCAGTTGGCGGGGTAACGGCCCACCAAAGCGACGATCAGTAGCCGACCTGAGAGGG TGACCGGCCACATTGGGGACCTGAGACACGGCCCA OTU268 (SEQ ID NO.: 103)GATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAGCGAAGCGCTGT TTTCAGAATCTTCGGAGGAAGAGGACAGTGACTGAGCGGCGGACGGGTGAGTAACGCGTG GGCAACCTGCCTCATACAGGGGGATAACAGTTAGAAATGACTGCTAATACCGCATAAGCG CACAGGACCGCATGGTGTAGTGTGAAAAACTCCGGTGGTATGAGATGGACCCGCGTCTGA TTAGGTAGTTGGTGGGGTAAAGGCCTACCAAGCCGACGATCAGTAGCCGACCTGAGACGG GTGACCGGCACATTGGGGACTGAGACACGGGCCCAA OTU269 (SEQ ID NO.: 104)GATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAACGAAGCATTTT AGATGAAGTTTTCGGATGGATTCTGAGATGACTGAGTGGCGGACGGGTGAGTAACACGTG GATAACCTGCCTCACACTGGGGGACGAACAGTTAGAAATAGACTGCTAATACCGCATAAG CGCACAGTACCGCATGGTACAGTGTGAAAAACTACCGGTGGTGTGAGATGGATCCGCGCT GATTAGTCCAGTTGGCGGGGTAACGGCCGACCAAAGCGACGATCAGTAGCCGACCTGAGA GGGTGACCGGCCGACAGTTGGGACTGAGACACGGCCCAA OTU277 (SEQ ID NO.: 105)GATGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAGCGAAGCACTAA GACGGATTTCTTTGGATTGAAGTCTTTGTGACTGAGCGGCGGACGGGTGAGTAACGCGTG GGTAACCTGCCTCATACAGGGGGATAACAGTTAGAAATGACTGCTAATACCGCATAAGCG CACAGGATCGCATGGTCTGGTGTGGAAAAACTCCGGTGGTATGAGATGGACCCGCGTCTG ATTAGCTAGTTGGAGGGGTAACGGCCCACCAAGGCGACGATCAGTAGCCGGCCTGAGAGG GTGAACGGCCACGATTGGGACTGAGACACGGCCCAG OTU279 (SEQ ID NO.: 106)GATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAACGAAGTTAGAC AGAGGAAGTTTTCGGATGGAATCGGTATAACTTAGTGGCGGACGGGTGAGTAACGCGTGG GAACCTGCCCTGTACCGGGGGAGTAACACTTAGAAATAGGTGCTAATACCGCATAAGCGC ACGGAACCGCATGGTTCTGTGTGAAAAACTACCGGTGGTACAGGATGGTCCCGCGTCTGA TTAGCCAGTTGGCAGGGTAACGGCCTACCAAAGCGACGATCAGTAGCCGGCCTGAGAGGG TGAACGGCCACATTGGGACTGAGACACGGCCCA OTU280 (SEQ ID NO.: 107)GATGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAACGCTTTGTAAA GGAGCTTGCTTCTTTACGAGGAGTGGCGAACGGGTGAGTAATACATAAGCAATCTGCCCA TCGGCCTGGGATAACAGTTGGAAACGACTGCTAATACCGGATAGGTTAGTTTCTGGCATC AGGGACTAATTAAAGTTGGGATACAACACGGATGGATGAGCTTATGGCGTATTAGCTAGT AGGTGAGGTAACGGCCCACCTAGGCGATGATACGTAGCCGACCTGAGAGGGTGACCGGCC ACATTGGGACTGAGACACGGCCCAA OTU281 (SEQ ID NO.: 39)GATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAACGAAGCGCTTC CGCCTGATTTTCTTCGGAGATGAAGGCGGCTGCGACTGAGTGGCGGACGGGTGAGTAACG CGTGGGCAACCTGCCTTGCACTGGGGGATAACAGCCAGAAATGGCTGCTAATACCGCATA AGACCGAAGCGCCGCATGGCGCTGCGGCCAAAGCCCCGGCGGTGCAAGATGGGCCCGCGT CTGATTAGGTAGTTGGCGGGGTAACGGCCCACCAAGCCGACGATCAGTAGCCGACCTGAG AGGGTGACCGGCCACATTGGGACTGAGACACGGCCCA OTU286 (SEQ ID NO.: 108)GATGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAGCGAAGCACTAA GACGGATTTCTTCGGATTGAAGTCTTTGTGACTGAGCGGCGGACGGGTGAGTAACGCGTG GGTAACCTGCCTCATACAGGGGGATAACAGTTAGAAATGACTGCTAATACCGCATAAGCG CACAGGATCGCATGGTCTGGTGTGAAAAACTCCGGTGGTATGAGATGGACCCGCGTCTGA TTAGCCAGTTGGCAGGGTAACGGCCTACCAAAGCGACGATCAGTAGCCGGCCTGAGAGGG TGAACGGCCACATTGGGACTGAGACACGGGCCCAA OTU287 (SEQ ID NO.: 109)GACGAACGCTGGCGGCGCGCCTAACACATGCAAGTCGAACGGACACATCC GACGGAATAGCTTGCTAGGAAGATGGATGTTGTTAGTGGCGGACGGGTGAGTAACACGTG AGCAACCTGCCTCGGAGTGGGGGACAACAGTTGGAAACGACTGCTAATACCGCATACGGT GGTCGGGGGACATCCCCTGGCCAAGAAAGGATTATATCCGCTCTGAGATGGGCTCGCGTC TGATTAGCTAGTTGGCGGGTAATGGCCCGACCGAAGGCAACGATCAGTAGCCGGACTGAG AGGTTGAACGGCCACATTGGGACTGAGACACGGCCCCAG OTU288 (SEQ ID NO.: 40)GATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAACGGAGTTATGC AGAGGAAGTTTTCGGATGGAATCGGCGTAACTTAGTGGCGGACGGGTGAGTAACGCGTGG GAAACCTGCCCTGTACCGGGGGATAACACTTAGAAATAGGTGCTAATACCGCATAAGCGC ACAGCTTCACATGAAGCAGTGTGAAAAACTCCGGTGGTACAGGATGGTCCCGCGTCTGAT TAGCCAGTTGGCAGGGTAACGGCCTACCAAAGCGACGATCAGTAGCCGGCCTGAGAGGGT GAACGGCCACATTGGGACTGAGACACGGCCCA OTU296 (SEQ ID NO.: 23)GATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAACGAAGCGCCTG GCCCCGACTTCTTCGGAACGAGGAGCCTTGCGACTGAGTGGCGGACGGGTGAGTAACGCG TGGGCAACCTGCCTTGCACTGGGGGATAACAGCCAGAAATGGCTGCTAATACCGCATAAG ACCGAAGCGCCGCATGGCGCAGCGGCCAAAGCCCCGGCGGTGCAAGATGGGCCCGCGTCT GATTAGGTAGTTGGCGGGGTAACGGCCCACCAAGCCGACGATCAGTAGCCGACCTGAGAG GGTGACCGGCCACATTGGGACTGAGACACGGCCCA OTU297 (SEQ ID NO.: 110)GATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAACGAAGCATCTT ATAGGAAGTTTTCGGATGGAATATGGGATGACTGAGTGGCGGACGGGTGAGTAACGCGTG GATAACCTGCCTCACACTGGGGGAGTAACAGTTAGAAATGGCTGCTAATACCCCACTAAG CGCACGGTACCGCATGGTACGGTGTGAAAAACCCAGGTGGTGTGAGATGGATCCGCGTCT GATTAGCCAGTTGGCGGGGTAACGGCCCGACCAAACGCGACGATCAGTAGCCGACCTGAG AGGGTGACCGGCCGACATTGGGACTGAGACACGGCCCA OTU303 (SEQ ID NO.: 111)GATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAACGAAGCATTTT AGATGAAGTTTTCGGATGGATTCTGAGATGACTGAGTGGCGGACGGGTGAGTAACACGTG GATAACCTGCCTCACACTGGGGGACAACAGTTAGAAATGACTGCTAATACCGCATAAGCG CACAGTACCGCATGGTACAGCGTGAAAAACTCCGGTGGTGTGAGATGGATCCGCGTCTGA TTAGCCAGTTGGCGGGGTAACGGCCCACCAAAGCGACGATCAGTAGCCGACCTGAGAGGG TGACCGGCACATTGGGGACTGAGACCACGGGCCCAA OTU304 (SEQ ID NO.: 112)ATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAACGGTAACAGGA AGCAGCTTGCTGCTTTGCTGACGAGTGGCGGACGGGTGAGTAATGTCTGGGAAACTGCCC GATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCGCAAGACCAAA GAGGGGGACCTTAGGGCCTCTTGCCATCGGATGTGCCCAGATGGGATTAGCTAGTAGGTG GGGTAAAGGCTCACCTAGGCGACGATCCCTAGCTGGTCTGAGAGGATGACCAGCCACACT GGAACTGAGA CACGGTCCAGOTU306 (SEQ ID NO.: 33)GATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAACGAAGCGACTT AACGGAAGTTTTCGGATGGAAGTTGAATTGACTGAGTGGCGGACGGGTGAGTAACGCGTG GGTAACCTGCCTTGTACTGGGGGACGAACAGTTAGAAATGACTGCTAATACCGCATAAGC GCACAGTATCGCATGATACAGTGTGAAAAACTCCGGTGGTACAAGATGGACCCGCGTCTG ATTAGCTAGTTGGTAAGGTAACGGCTTACCAAGGCGACGATCAGTAGCCGACCTGAGAGG GTGACCGGCCACATTGGGACTGAGACACGGCCCA OTU307 (SEQ ID NO.: 113)GATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAACGAAGCATCTT ATAGGAAGTTTTCGGATGGAATATGGGATGACTGAGTGGCGGACGGGTGAGTAACGCGTG GAGTAACCTGCCTCACACTGGGGGATAACAGTTAGAAATGGCTGCTAATACCCCATAAGC GCACAGTACCGCATGGTACGGTGTGAAAAACCCAGGTGGTGTGAGATGGATCCGCGTCTG ATTAGCCAGTTGGCGGGTAACGGCCGACCAAAGCGACGATCAGTAGCCGACCTGAGAGGG TGACCGGCACGATTGGGACCTGAGACACGGGCCCA OTU312 (SEQ ID NO.: 114)GATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAACGAAGTTATAT CGAGGAAGTTTTCGGATGGAATCAGTATAACTTAGTGGCGGACGGGTGAGTAACGCGTGG GAAACCTGCCCTGTACCGGGGGATAACACTTAGAAATAGGTGCTAATACCGCATAAGCGC ACAGCTTCACATGAAGCAGTGTGAAAAACTCCGGTGGTATGAGATGGACCCGCGTCTGAT TAGCTAGTTGGAGGGGTAACGGCCCACCAAGGCGACGATCAGTAGCCGGCCTGAGAGGGT GAACGGCCACATTGGGACTGAGACACGGCCCAG OTU313 (SEQ ID NO.: 115)GATGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAACGCGGGCAGCA ATGCCCGAGTGGCGAACGGGTGAGTAATACATAAGTAACCTGCCCTTTACAGGGGGATAA CTATTGGAAACGATAGCTAAGACCGCATAGGTAAAGATACCGCATGGTAAGTTTATTAAA GTGCCAAGGCACTGGTAGAGGATGGACTTATGGCGCATTAGCTAGTTGGTGAGGTAACGG CTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGACCGGCCACACTGGGACTGAG ACACGGCCCA A OTU314(SEQ ID NO.: 31) GATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAGCGAAGCGCTGTTTTCAGAATC TTCGGAGGAAGAGGACAGTGACTGAGCGGCGGACGGGTGAGTAACGCGTG GGCAACCTGCCTCATACAGGGGGATAACAGTTAGAAATGACTGCTAATACCGCATAAGCG CACAGGACCGCATGGTGTAGTGTGAAAAACTCCGGTGGTATGAGATGGACCCGCGTCTGA TTAGGTAGTTGGTGGGGTAAGGCCGTACCAAGCCGACGATCAGTAGCCGACCTGAGAGGG TGACCGGCCACATTGGGGACTGAGACACGGCCCA OTU319 (SEQ ID NO.: 116)GATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAACGAAGTTAGAC AGAGGAAGTTTTCGGATGGAATCGGTATAACTTAGTGGCGGACGGGTGAGTAACGCGTGG GAAACCTGCCCTGTACCGGGGGATAACACTTAGAAATGACTGCTAATACCGCATAAGCGC ACAGTACCGCATGGTACAGTGTGAAAAACTCCGGTGGTGTGAGATGGATCCGCGTCTGAT TAGCCAGTTGGCGGGGTAACGGCCCACCAAAGCGACGATCAGTAGCCGACCTGAGAGGGT GACCGGCACATTGGGACTGAGACACGGCCCAA OTU326 (SEQ ID NO.: 117)GATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAACGAAGCAATTA AAATGAAGTTTTCGGATGGATTTTTGATTGACTGAGTGGCGGACGGGTGAGTAACGCGTG GATAACCTGCCTCACACTGGGGGATAACAGTTAGAAATGACTGCTAATACCGCATAAGCG CACAGCTTCACATGAAGCAGTGTGAAAAACTCCGGTGGTACAGGATGGTCCCGCGTCTGA TTAGCCAGTTGGCAGGGTAACGGCCTACCAAAGCGACGATCAGTAGCCGGCCTGAGAGGG TGAACGGCCACATTGGGACTGAGACACGGCCCAA OTU328 (SEQ ID NO.: 118)GATGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAACGGAGTGCCTT AGAAAGAGGATTCGTCCAATTGATAAGGTTACTTAGTGGCGGACGGGTGAGTAACGCGTG AGGAACCTGCCTCGGAGTGGGGAATAACAGACCGAAAGGTCTGCTAATACCGCATGATGC AGTTGGACCGCATGGTCCTGACTGCCAAAGATTTATCGCTCTGAGATGGCCTCGCGTCTG ATTAGCTTGTTGGCGGGGTAATGGCCCACCAAGGCGACGATCAGTAGCCGGACTGAGAGG TTGGCCGGCCACATTGGGACTGAGACACGGCCCA OTU333 (SEQ ID NO.: 119)GATGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAACGGAGTGCTCA TGACAGAGGATTCGTCCAATGGAGTGAGTTACTTAGTGGCGGACGGGTGAGTAACGCGTG AGTAACCTGCCTTGGAGTGGGGAATAACAGGTGGAAACATCTGCTAATACCGCATGATGC AGTTGGGTCGCATGGCTCTGACTGCCAAAGATTTATCGCTCTGAGATGGACTCGCGTCTG ATTAGCTGGTTGGCGGGTAACGGCCACCAAGGCGACGATCAGTAGCCGGACTGAGAGGTT GGCCGGCCACATTGGGACTGAGACACGGCCCAG OTU334 (SEQ ID NO.: 41)GATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAACGAAGCATCCC GATAGAAGTTTTCGGATGGAATATGGGATGACTGAGTGGCGGACGGGTGAGTAACGCGTG GATAACCTGCCTCACACTGGGGGATAACAGTTAGAAATGGCTGCTAATACCGCATAAGCG CACAGTACCGCATGGTACGGTGTGAAAAACCCAGGTGGTGTGAGATGGATCCGCGTCTGA TTAGCCAGTTGGCGGGGTAACGGCCCACCAAAGCGACGATCAGTAGCCGACCTGAGAGGG TGACCGGCCACATTGGGGACTGAGACACGGCCCA OTU337 (SEQ ID NO.: 30)GACGAACGCTGGCGGCGCGCCTAACACATGCAAGTCGAACGGAGCTTACG TTTTGAAGTTTTCGGATGGATGAATGTAAGCTTAGTGGCGGACGGGTGAGTAACACGTGA GCAACCTGCCTTTCAGAGGGGGATAACAGCCGGAAACGGCTGCTAATACCGCATGATGTT GCGGGGGCACATGCCCCTGCAACCAAAGGAGCAATCCGCTGAAAGATGGGCTCGCGTCCG ATTAGCCAGTTGGCGGGGTAACGGCCCACCAAAGCGACGATCGGTAGCCGGACTGAGAGG TTGAACGGCCACATTGGGACTGAGACACGGCCCAG OTU339 (SEQ ID NO.: 120)GATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAACGAAGCAATTA AAATGAAGTTTTCGGATGGATTTTTGATTGACTGAGTGGCGGACGGGTGAGTAACGCGTG GATAACCTGCCTCACACTGGGGATAACAGTTAGAAATGACTGCTAATACCGCATAAGCGC ACAGTACCGCATGGTACGGTGTGAAAAACTCCGGTGGTGTGAGATGGATCCGCGTCTGAT TAGCCAGTTGCGGGGTAACGGCCCGACCAAGCGACGATCAGTAGCCGACCGTGAGAGGTG ACCGGCCCACATTGGGACTGAGACACGGCCCAA OTU340 (SEQ ID NO.: 121)GACGAACGCTGGCGGCGCGCCTAACACATGCAAGTCGAACGGAGTTGTGT TGAAAGCTTGCTGGATATACAACTTAGTGGCGGACGGGTGAGTAACACGTGAGTAACCTG CCTCTCAGAGTGGAATAACGTTTGGAAACGAACGCTAATACCGCATAACGTGAGAAGAGG GCATCCTCTTTTTACCAAAGATTTATCGCTGAGAGATGGGCTCGCGGCCGATTAGGTAGT TGGTGAGATAACAGCCCACCAAGCCGACGATCGGTAGCCGGACTGAGAGGTTGATCGGCC ACATTGGGACTGAGACACGGCCCAG OTU353 (SEQ ID NO.: 122)GATGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAGCGAAGCACCTT GACGGATTCTTCGGATTGAAGCCTTGGTGACTGAGCGGCGGACGGGTGAGTAACGCGTGG GTAACCTGCCTCATACAGGGGGGATAAACAGTTAGAAATGACTGCTAATACCGCATAAGC GCACAGGACCGCATGGTCTGGTGTGAAAAACTCCGGTGGTATGAGATGGACCCGCGTCTG ATTAGCTAGTTGGAGGGGTAACGGCCCACCAAGGCGACGATCAGTAGCCGGCCTGAGAGG GTGAACGGCCACATTGGGACTGAGGACACGGCCCA OTU359 (SEQ ID NO.: 42)GATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAACGAAGTTATAT CGAGGAAGTTTTCGGATGGAATCAGTATAACTTAGTGGCGGACGGGTGAGTAACGCGTGG GAAACCTGCCCTGTACCGGGGGATAACACTTAGAAATAGGTGCTAATACCGCATAAGCGC ACAGCTTCACATGAAAGCAGTGTGAAAAACTCCGGTGGTACAGGATGGTCCCGCGTCTGA TTAGCCAGTTGGCAGGGTAACGGCCTACCAAAGCGACGATCAGTAGCCGGCCTGGAGAGG GTGAACGGCCACATTGGGACTGAGACACGGCCCG OTU362 (SEQ ID NO.: 43)GATGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAGCGAAGCGGTTT CGATGAAGTTTTCGGATGGATTTGAAATCGACTTAGCGGCGGACGGGTGAGTAACGCGTG GGTAACCTGCCTTACACTGGGGATAACAGTTAGAAATGACTGCTAATACCGCATAAGCGC ACAGGGCCGCATGGTCCGGTGTGAAAACTCCGGTGGTGTAAGATGGACCCGCGTCTGATT AGGTAGTTGGTGGGTAACGGCCCACCAAGCCGACGATCAGTAGCCGACCTGAGAGGGTGA CCGGCCACATTGGGACTGAGACACGGCCCAA OTU367 (SEQ ID NO.: 44)GATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAACGAAGTTACAC AGAGGAAGTTTTCGGATGGAATCGGTATAACTTAGTGGCGGACGGGTGAGTAACGCGTGG GAAACCCGCCCTGTACCGGGGGATAACACTTAGAAATAGGTGCTAATACCGCATAAGCGC ACAGCTTCACATGAAGCAGTGTGAAAACTCCGGTAGGTACAGGATGGTCCCGCGTCTGAT TAGCCAGTTGGCAGGGTAACGGCCTACCAAAGCGACGATCAGTAGCCGGCCTGAGAGGGT CAACGGCCACATTGGGACTGAGACACGGCCCAA

What is claimed is:
 1. A method of treating autoimmune disease in asubject, the method comprising administering a pharmaceuticalcomposition comprising a purified bacterial mixture consisting ofbacteria comprising 16S rDNA sequences of at least 95% homology to SEQID NO:22, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:31, SEQ IDNO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:40, SEQ ID NO:41, and SEQID NO:42 to the subject in an amount sufficient to treat autoimmunedisease.
 2. The method of claim 1, wherein the autoimmune disease isorgan transplant rejection, inflammatory bowel disease (IBD), ulcerativecolitis, pouchitis, Crohn's disease, sprue, rheumatoid arthritis, Type 1diabetes, graft versus host disease, or multiple sclerosis.
 3. Themethod of claim 2, wherein the autoimmune disease is inflammatory boweldisease (IBD), ulcerative colitis, pouchitis, or Crohn's disease.
 4. Themethod of claim 1, wherein the bacteria are human-derived bacteria. 5.The method of claim 1, wherein the bacteria are isolated from achloroform-treated fecal sample.
 6. The method of claim 1, wherein thebacteria are isolated from a heat-treated fecal sample.
 7. The method ofclaim 1, wherein at least a portion of the bacteria are in spore-form.8. The method of claim 1, further comprising a pharmaceuticallyacceptable excipient.
 9. The method of claim 1, wherein thepharmaceutical composition is formulated for oral administration. 10.The method of claim 1, wherein the pharmaceutical composition comprisesone or more enteric polymers.